Challenge of Naïve and Vaccinated Pigs with a Vaccine-Derived Recombinant Porcine Reproductive and Respiratory Syndrome Virus 1 Strain (Horsens Strain).

In July 2019, a vaccine-derived recombinant Porcine reproductive and respiratory syndrome virus 1 strain (PRRSV-1) (Horsens strain) infected more than 40 Danish sow herds, resulting in severe losses. In the present study, the pathogenicity of the recombinant Horsens strain was assessed and compared to a reference PRRSV-1 strain using a well-characterized experimental model in young SPF pigs. Furthermore, the efficacies of three different PRRSV-1 MLV vaccines to protect pigs against challenge with the recombinant strain were assessed. Following challenge, the unvaccinated pigs challenged with the Horsens strain had significant increased viral load in serum compared to all other groups. No macroscopic changes were observed at necropsy, but tissue from the lungs and tonsils from almost all pigs were PRRSV-positive. The viral load in serum was lower in all vaccinated groups compared to the unvaccinated group challenged with the Horsens strain, and only small differences were seen among the vaccinated groups. The findings in the present study, combined with two other recent reports, indicate that this recombinant "Horsens" strain indeed is capable of inducing infection in growing pigs as well as in pregnant sows that is comparable to or even exceeding those induced by typical PRRSV-1, subtype 1 strains. However, absence of notable clinical signs and lack of significant macroscopic changes indicate that this strain is less virulent than previously characterized highly virulent PRRSV-1 strains.

Assessment of the Impact of the Recombinant Porcine Reproductive and Respiratory Syndrome Virus Horsens Strain on the Reproductive Performance in Pregnant Sows.

This study assessed the impact of a PRRSV (porcine reproductive and respiratory syndrome virus) recombinant strain (Horsens strain) on the reproductive performance of naïve pregnant sows in the last third of gestation. Fifteen sows were included: four negative reproductive controls (NTX), five infected with a PRRSV-1 field strain (Olot/91, T01), and six infected with the recombinant PRRSV-1 strain (Horsens strain, T02). Piglets were monitored until weaning. Reproductive performance was the primary variable. In sows, viremia and nasal shedding (T01 and T02 groups), and, in piglets, viral load in blood and in lungs, as well as macroscopic lung lesions (T01 and T02 groups), were the secondary variables. The reproductive performance results were numerically different between the two challenged groups. Moreover, viral loads in blood were 1.83 × 106 ± 9.05 × 106 copies/mL at farrowing, 1.05 × 107 ± 2.21 × 107 copies/mL at weaning from piglets born from T01 animals and 1.64 × 103 ± 7.62 × 103 copies/mL at farrowing, 1.95 × 103 ± 1.17 × 104 copies/mL at weaning from piglets born from T02 sows. Overall, 68.8% of T01 piglets and 38.1% of T02 piglets presented mild lung lesions. In conclusion, the results suggest that Horsens strain is less virulent than the field strain Olot/91 under these experimental conditions.

A Triple Amino Acid Substitution at Position 88/94/95 in Glycoprotein GP2a of Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV1) Is Responsible for Adaptation to MARC-145 Cells.

The Meat Animal Research Center-145 (MARC-145) cell line has been proven to be valuable for viral attenuation regarding vaccine development and production. Cell-adaptation is necessary for the efficient replication of porcine reproductive and respiratory syndrome virus (PRRSV) in these cells. Multiple sequence analysis revealed consistent amino acid substitutions in GP2a (V88F, M94I, F95L) of MARC-145 cell-adapted strains. To investigate the putative effect of these substitutions, mutations at either position 88, 94, 95, and their combinations were introduced into two PRRSV1 (13V091 and IVI-1173) infectious clones followed by the recovery of viable recombinants. When comparing the replication kinetics in MARC-145 cells, a strongly positive effect on the growth characteristics of the 13V091 strain (+2.1 log10) and the IVI-1173 strain (+1.7 log10) compared to wild-type (WT) virus was only observed upon triple amino acid substitution at positions 88 (V88F), 94 (M94I), and 95 (F95L) of GP2a, suggesting that the triple mutation is a determining factor in PRRSV1 adaptation to MARC-145 cells.

Pathogenicity of three genetically diverse strains of PRRSV Type 1 in specific pathogen free pigs.

Studies from Eastern European countries proved that porcine reproductive and respiratory syndrome virus Type 1 (PRRSV-1) harbours high genetic diversity and that genetically divergent subtypes 2-4 circulate in this area. In the present study, we compared the pathogenicity of two different PRRSV-1 subtype 2 strains and a strain representing PRRSV-1 subtype 1. Four groups of 8-week-old specific pathogen free pigs were either infected with subtype 2 strain ILI6, subtype 2 strain or BOR59, subtype 1 strain 18794, or mock inoculated. The most pronounced clinical signs were observed in pigs infected with BOR59. Pigs from both subtype 2 strain infected groups exhibited significantly elevated mean body temperatures on DPI 2 compared to the other two groups, the difference remaining significant up to DPI 13 for the BOR59 group, only. The pigs in the latter group also displayed significantly highest levels of early viremia together with the most rapid APP response. Overall, the results indicated that BOR59 strain can be considered a highly pathogenic strain, similarly to subtype 3 strains Lena and SU1-bel, while the virulence of the other subtype 2 strain ILI6 was intermediate between BOR59 and subtype 1 strain.

The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses.

The porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of one of the most economically important diseases affecting swine worldwide. Efforts to develop a next-generation vaccine have largely focused on envelope glycoproteins to target virus-neutralizing antibody responses. However, these approaches have failed to demonstrate the necessary efficacy to progress toward market. T cells are crucial to the control of many viruses through cytolysis and cytokine secretion. Since control of PRRSV infection is not dependent on the development of neutralizing antibodies, it has been proposed that T cell-mediated immunity plays a key role. Therefore, we hypothesized that conserved T cell antigens represent prime candidates for the development a novel PRRS vaccine. Antigens were identified by screening a proteome-wide synthetic peptide library with T cells from cohorts of pigs rendered immune by experimental infections with a closely related (subtype 1) or divergent (subtype 3) PRRSV-1 strain. Dominant T cell IFN-γ responses were directed against the non-structural protein 5 (NSP5), and to a lesser extent, the matrix (M) protein. The majority of NSP5-specific CD8 T cells and M-specific CD4 T cells expressed a putative effector memory phenotype and were polyfunctional as assessed by coexpression of TNF-α and mobilization of the cytotoxic degranulation marker CD107a. Both antigens were generally well conserved among strains of both PRRSV genotypes. Thus, M and NSP5 represent attractive vaccine candidate T cell antigens, which should be evaluated further in the context of PRRSV vaccine development.

Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain.

Stable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p < 0.05) in the control group (7.6 ± 1.7 days) compared to the immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (p < 0.05) of AUC values of nasal shedding (control: 14.6, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (p < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe.

Different clinical, virological, serological and tissue tropism outcomes of two new and one old Belgian type 1 subtype 1 porcine reproductive and respiratory virus (PRRSV) isolates.

In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed to identify their origin. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of sialoadhesin(-) infected cells/mm(2) in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for sialoadhesin(-) cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication and shedding, but a decreased replication in lymphoid tissues compared to 07V063.

Genetic dissection of complete genomes of Type 2 PRRS viruses isolated in Denmark over a period of 15 years.

Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was first detected in Europe in 1996 co-incident with the introduction of a live attenuated vaccine. Since then, only limited ORF5 and ORF7 sequences of Type 2 PRRS viruses have been reported throughout Europe. In the present study, the genetic and antigenic diversity of 11 complete genomes and 49 ORF5 and 55 ORF7 nucleotide sequences obtained from 57 viruses in Denmark from 2003 to 2012 were examined. The genetic identity of the 11 complete genomes to the vaccine strain (Ingelvac PRRS MLV) ranged between 93.6 and 99.6% while the 49 ORF5 sequences examined were 94.0-99.8% identical to the vaccine strain. Among the Danish sequences, the pairwise nucleotide identity was 90.9-100% and 93.0-100.0% for ORF5 and ORF7, respectively. Analysis of the genetic region encoding NSP2 revealed high diversity among the Danish viruses with an 86.6-98.9% range in similarity. Furthermore, several of the sequenced viruses harbored deletions in the NSP2 coding region. Phylogenetic analysis in a global Type 2 PRRSV framework classified all Danish isolates to a single cluster (sub-lineage 5.1) which comprised strains closely-related to the Type 2 prototype isolate VR2332.

Genetic and antigenic characterization of complete genomes of Type 1 Porcine Reproductive and Respiratory Syndrome viruses (PRRSV) isolated in Denmark over a period of 10 years.

Porcine Reproductive and Respiratory Syndrome (PRRS) caused by the PRRS virus (PRRSV) is considered one of the most devastating swine diseases worldwide. PRRS viruses are divided into two major genotypes, Type 1 and Type 2, with pronounced diversity between and within the genotypes. In Denmark more than 50% of the herds are infected with Type 1 and/or Type 2 PRRSV. The main objective of this study was to examine the genetic diversity and drift of Type 1 viruses in a population with limited introduction of new animals and semen. A total of 43 ORF5 and 42 ORF7 nucleotide sequences were obtained from viruses collected from 2003 to February 2013. Phylogenetic analysis of ORF5 nucleotide sequences showed that the Danish isolates formed two major clusters within the subtype 1. The nucleotide identity to the subtype 1 protogenotype Lelystad virus (LV) spanned 84.9-98.8% for ORF5 and 90.7-100% for ORF7. Among the Danish viruses the pairwise nucleotide identities in ORF5 and ORF7 were 81.2-100% and 88.9-100%, respectively. Sequencing of the complete genomes, including the 5'- and 3'-end nucleotides, of 8 Danish PRRSV Type 1 showed that the genome lengths differed from 14,876 to 15,098 nucleotides and the pairwise nucleotide identity among the Danish viruses was 86.5-97.3% and the identity to LV was 88.7-97.9%. The study strongly indicated that there have been at least two independent introductions of Type 1 PRRSV in Denmark and analysis of the full genomes revealed a significant drift in several regions of the virus.

A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2.

PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.