WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

Evolutionary Dynamics of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Whole-Genome Analysis.

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen in the swine industry, is a genetically highly diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus are not yet fully understood. In this study, we performed an integrated analysis of all available whole-genome sequences of type 2 PRRSV (n = 901) to reveal its evolutionary dynamics. The results showed that there were three distinct phylogenetic lineages of PRRSV in their distribution patterns. We identified that sublineage 2.7 (L2.7), associated with a NADC30 cluster, had the highest substitution rate and higher viral genetic diversity, and inter-lineage recombination is observed more frequently in L2.7 PRRSV compared to other sublineages. Most inter-lineage recombination events detected are observed between L2.7 PRRSVs (as major parents) and L3.4 (a JXA1-R-related cluster)/L3.7 (a WUH3-related cluster) PRRSVs (as minor parents). Moreover, the recombination hotspots are located in the structural protein gene ORF2 and ORF4, or in the non-structural protein gene nsp7. In addition, a GM2-related cluster, L3.2, shows inconsistent recombination modes compared to those of L2.7, suggesting that it may have undergone extensive and unique recombination in their evolutionary history. We also identified several amino acids under positive selection in GP2, GP4 and GP5, the major glycoproteins of PRRSV, showing the driving force behind adaptive evolution. Taken together, our results provide new insights into the evolutionary dynamics of PPRSV that contribute to our understanding of the critical factors involved in its evolution and guide future efforts to develop effective preventive measures against PRRSV.

Porcine Reproductive and Respiratory Syndrome (PRRS) Epidemiology in an Integrated Pig Company of Northern Italy: A Multilevel Threat Requiring Multilevel Interventions.

Porcine reproductive and respiratory syndrome (PRRS) is probably the most relevant viral disease affecting pig farming. Despite the remarkable efforts paid in terms of vaccination administration and biosecurity, eradication and long-term control have often been frustrated. Unfortunately, few studies are currently available that objectively link, using a formal statistical approach, viral molecular epidemiology to the risk factors determining the observed scenario. The purpose of the present study is to contribute to filling this knowledge gap taking advantage of the advancements in the field of phylodynamics. Approximately one-thousand ORF7 sequences were obtained from strains collected between 2004 and 2021 from the largest Italian pig company, which implements strict compartmentalization among independent three-sites (i.e., sow herds, nurseries and finishing units) pig flows. The history and dynamics of the viral population and its evolution over time were reconstructed and linked to managerial choices. The viral fluxes within and among independent pig flows were evaluated, and the contribution of other integrated pig companies and rurally risen pigs in mediating such spreading was investigated. Moreover, viral circulation in Northern Italy was reconstructed using a continuous phylogeographic approach, and the impact of several environmental features on PRRSV strain persistence and spreading velocity was assessed. The results demonstrate that PRRSV epidemiology is shaped by a multitude of factors, including pig herd management (e.g., immunization strategy), implementation of strict-independent pig flows, and environmental features (e.g., climate, altitude, pig density, road density, etc.) among the others. Small farms and rurally raised animals also emerged as a potential threat for larger, integrated companies. These pieces of evidence suggest that none of the implemented measures can be considered effective alone, and a multidimensional approach, ranging from individual herd management to collaboration and information sharing among different companies, is mandatory for effective infection control.

Temporal lineage dynamics of the ORF5 gene of porcine reproductive and respiratory syndrome virus in Korea in 2014-2019.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the Korean swine industry. Despite efforts including improved biosecurity and vaccination protocols, the virus continues to circulate and evolve. Based on phylogenetic analysis of open reading frame 5 (ORF5), Korean PRRSVs are known to form not only globally circulating lineages but also country-specific lineages (Lin Kor A, B, and C). To understand the recent epidemiological status of PRRSV in Korea, a total of 1349 ORF5 sequences of Korean PRRSV isolates from 2014 to 2019 were analyzed. Phylogenetic analysis was conducted using the maximum-likelihood method, and temporal changes in the relative prevalence of lineages were investigated. The analysis showed that PRRSV1 and PRRSV2 were both highly prevalent throughout the years examined. Among the PRRSV1 isolates, subgroup A (90.1%) and vaccine-like subgroup C (9.0%) composed most of the population. For PRRSV2 isolates, vaccine-like lineage 5 (36.3%) was dominant, followed by Lin Kor B (25.9%), Kor C (16.6%), lineage 1 (11.6%), and Kor A (9.1%). The PRRSV2 lineage 1 population increased from 2014 (1.8%) to 2019 (29.6%) in Korea due to the continual spread of sublineage 1.8 (NADC30-like) and introduction of sublineage 1.6 into the country. Additional genetic analysis, including analysis of non synonymous and synonymous mutations, revealed evidence of diversification and positive selection in immunologically important regions of the genome, suggesting that current vaccination is failing and promoting immune-mediated selection. Overall, these findings provide insights into the epidemiological and evolutionary dynamics of cocirculating viral lineages, and constant surveillance of PRRSV occurrence is needed.

Efficacy of a live attenuated highly pathogenic PRRSV vaccine against a NADC30-like strain challenge: implications for ADE of PRRSV.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection can cause severe reproductive failure in sows and respiratory distress in pigs of all ages, leading to major economic losses. To date, there are still no effective strategies to prevent and control PRRSV. Antibody-dependent enhancement (ADE), a phenomenon in which preexisting non-neutralizing antibodies or sub-neutralizing antibodies facilitate virus entry and replication, may be a significant obstacle in the development of effective vaccines for many viruses, including PRRSV. However, the contribution of ADE to PRRSV infection remains controversial, especially in vivo. Whether attenuated PRRSV vaccines prevent or worsen subsequent disease in pigs infected by novel PRRSV strains requires more research. In the present study, in vivo experiments were conducted to evaluate ADE under different immune statuses, which were produced by waiting different lengths of time after vaccination with a commercially available attenuated highly pathogenic PRRSV (HP-PRRSV) vaccine (JXA1-R) before challenging the pigs with a novel heterologous NADC30-like strain. RESULTS: Piglets that were vaccinated before being challenged with PRRSV exhibited lower mortality rates, lower body temperatures, higher bodyweight gain, and lower viremia. These results demonstrate that vaccination with JXA1-R alleviated the clinical signs of PRRSV infection in all vaccinated groups. CONCLUSIONS: The obtained data indicate that the attenuated vaccine test here provided partial protection against the NADC30-like strain HNhx. No signs of enhanced PRRSV infection were observed under the applied experimental conditions. Our results provide some insight into the molecular mechanisms underlying vaccine-induced protection or enhancement in PRRSV.

Evolutionary Patterns of Codon Usage in Major Lineages of Porcine Reproductive and Respiratory Syndrome Virus in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is economically important and characterized by its extensive variation. The codon usage patterns and their influence on viral evolution and host adaptation among different PRRSV strains remain largely unknown. Here, the codon usage of ORF5 genes from lineages 1, 3, 5, and 8, and MLV strains of type 2 PRRSV in China was analyzed. A compositional property analysis of ORF5 genes revealed that nucleotide C is most frequently used at the third position of codons, accompanied by rich GC3s. The effective number of codon (ENC) and codon pair bias (CPB) values indicate that all ORF5 genes have low codon bias and the differences in CPB scores among four lineages are almost not significant. When compared with host codon usage patterns, lineage 1 strains show higher CAI and SiD values, with a high similarity to pig, which might relate to its predominant epidemic propensity in the field. The CAI, RCDI, and SiD values of ORF5 genes from different passages of MLV JXA1R indicate no relation between attenuation and CPB or codon adaptation decrease during serial passage on non-host cells. These findings provide a novel way of understanding the PRRSV's evolution, related to viral survival, host adaptation, and virulence.

Analysis of ORF5 sequences of Porcine Reproductive and Respiratory Syndrome virus (PRRSV) circulating within swine farms in Costa Rica.

BACKGROUND: Worldwide, Porcine Reproductive and Respiratory Syndrome (PRRS) is among the diseases that cause the highest economic impact in modern pig production. PRRS was first detected in Costa Rica in 1996 and has since then severely affected the local swine industry. Studies of the molecular characterization of circulating strains, correlation with clinical records, and associations with pathogens associated with Porcine Respiratory Disease Complex (PRDC) have not been done in Costa Rica. RESULTS: Sequencing and phylogenetic analysis of ORF5 proved that PRRSV-2 was the only species detected in all locations analyzed. These sequences were grouped into three clusters. When comparing samples from San Jose, Alejuela, and Puntarenas to historical isolates of the previously described lineages (1 to 9), it has been shown that these were closely related to each other and belonged to Lineage 5, along with the samples from Heredia. Intriguingly, samples from Cartago clustered in a separate clade, phylogenetically related to Lineage 1. Epitope analysis conducted on the GP5 sequence of field isolates from Costa Rica revealed seven peptides with at least 80% amino acid sequence identity with previously described and experimentally validated immunogenic regions. Previously described epitopes A, B, and C, were detected in the Santa Barbara-Heredia isolate. CONCLUSIONS: Our data suggest that the virus has three distinct origins or introductions to the country. Future studies will elucidate how recently introduced vaccines will shape the evolutionary change of circulating field strains.

Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Development of a bead-based assay for detection and differentiation of field strains and four vaccine strains of type 2 porcine reproductive and respiratory syndrome virus (PRRSV-2) in the USA.

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most economically devastating diseases in swine population in the United States of America. Due to high mutation rate of the PRRS virus (PRRSV) genome, it is difficult to develop an accurate diagnostic assay with high strain coverage. Differentiation of field strains from the four vaccines that have been used in the USA, namely Ingelvac PRRS MLV, Ingelvac ATP, Fostera PRRS and Prime Pac PRRS, adds an additional challenge. It is difficult to use current real-time PCR systems to detect and differentiate the field strains from the vaccine strains. Luminex xTAG technology allows us to detect more molecular targets in a single reaction with a cost similar to a single real-time PCR reaction. By analysing all available 678 type 2 PRRSV (PRRSV-2) complete genome sequences, including the 4 vaccine strains, two pairs of detection primers were designed targeting the conserved regions of ORF4-ORF7, with strain coverage of 98.8% (670/678) based on in silico analysis. The virus strains sharing ≥98% identity of the complete genomes with the vaccine strains were considered vaccine or vaccine-like strains. One pair of primers for each vaccine strain were designed targeting the nsp2 region. In silico analysis showed the assay matched 94.7% (54/57) of Ingelvac PRRS® MLV (MLV) strain and the MLV-like strains, and 100% of the other three vaccine strains. Analytical sensitivity of the Luminex assay was one to two logs lower than that of the reverse transcription real-time PCR assay. Evaluated with 417 PRRSV-2 positive clinical samples, 95% were detected by the Luminex assay. Compared to ORF5 sequencing results, the Luminex assay detected 92.4% (73/79) of MLV strains, 78.3% (18/23) of Fostera strains and 50% (2/4) of ATP strains. None of the 472 samples were the Prime Pac strain tested by either ORF5 sequencing or the Luminex assay.

Porcine reproductive and respiratory syndrome virus dissemination across pig production systems in the United States.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains widespread in the North American pig population. Despite improvements in virus characterization, it is unclear whether PRRSV infections are a product of viral circulation within production systems (local) or across production systems (external). Here, we examined the local and external dissemination dynamics of PRRSV and the processes facilitating its spread in three production systems. Overall, PRRSV genetic diversity has declined since 2018, while phylodynamic results support frequent external transmission. We found that PRRSV dissemination predominantly occurred mostly through transmission between farms of different production companies for several months, especially from November until May, a timeframe already established as PRRSV season. Although local PRRSV dissemination occurred mainly through regular pig flow (from sow to nursery and then to finisher farms), an important flux of PRRSV dissemination also occurred in the opposite direction, from finisher to sow and nursery farms, highlighting the importance of downstream farms as sources of the virus. Our results also showed that farms with pig densities of 500 to 1,000 pig/km2 and farms located at a range within 0.5 km and 0.7 km from major roads were more likely to be infected by PRRSV, whereas farms at an elevation of 41 to 61 meters and surrounded by denser vegetation were less likely to be infected, indicating their role as dissemination barriers. In conclusion, our results demonstrate that external dissemination was intense, and reinforce the importance of farm proximity on PRRSV spread. Thus, consideration of farm location, geographic characteristics and animal densities across production systems may help to forecast PRRSV collateral dissemination.

Genetic characterization of a novel recombined porcine reproductive and respiratory syndrome virus 2 among Nadc30-like, Jxa1-like and TJ-like strains.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating viral diseases in the global pig industry, including China. Recently, we successfully isolated a porcine reproductive and respiratory syndrome virus (PRRSV) from lung tissue and peripheral blood of piglets at a farm from Dujiangyan in Sichuan, China, and named it the DJY-19 strain. The full-length genome sequence of DJY-19 shared 86.8%-94.1% nucleotide similarity with NADC30-like and NADC30 PRRSV strains. We compared the open reading frame (ORF) 5 gene of DJY-19 with 34 PRRSV strains from Genbank. Phylogenetic analysis showed that DJY-19 clustered with NADC30 strains, characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. The results of homology analysis showed that the homology between DJY-19 and NADC30 (JN654459.1) strains was the highest (95.9%), whereas homology with other domestic strains was lower (80.9%-92.6%). Furthermore, we identified four recombination breakpoints in the DJY-19 genome; they separated the DJY-19 genome into four regions. The 8106-9128 nucleotide (nt) region of DIY-19 was highly similar to the TJ strain, and the 12106-12580 nt region of DIY-19 was highly similar to the JXA1-R strain. Our findings demonstrate that DJY-19 arose from the recombination of North America NADC30 strain and TJ strain and JXA1-R in China. The application of multiple attenuated vaccine strains has led to complex recombination of PRRSV strains in China. This study provides a theoretical basis for making a more reasonable PRRS virus control and prevention strategy.

Histopathological Lesions Accompanied with First-Time Isolation of a PRRSV-2 Strain in Greece.

Genotype 2 strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) have been reported sporadically in Europe. Even if, PRRSV-2 reported to be genetically homogenous in Europe due to the introduction of an MLV vaccine strain, independent introductions of PRRSV-2 field strains have been reported. The aim of the present study was to report the complete genome sequence and evaluate the histopathological lesions of a PRRSV-2 strain, isolated for the first time in Greece. During a routine blood sampling in a commercial pig farm, the results revealed positive samples in weaners of 40-60 days for the PRRSV-2, using real-time polymerase chain reaction. The clinical picture was characterized from respiratory symptoms in weaners, as well as coughing and poor performance at finishing stage and less than 3% mortality rate from weaning stage to finishing stage. The use of ORF5 for PRRSV phylogenetic analysis of the isolated PRRSV strain, named "x1544-1 strain", was successfully determined, belonging to the genotype PRRSV-2. Comparison of the obtained sequence revealed nucleotide sequence identity >98% with PRRSV-2 strain VR2332 and other related strains from Denmark and China. The histopathological evaluation revealed diffuse interstitial pneumonia, multifocal interstitial nephritis, while in the lymphoid organs, follicular and paracortical hyperplasia, coexisting with necrosis and depletion of germ cells were detected. The results of current study undersign the importance for veterinary practitioners to have up-to-date access to phylogenetic data linked to phenotypic information to follow-up the control and prevention strategies against PRRSV.

Mir-331-3p Inhibits PRRSV-2 Replication and Lung Injury by Targeting PRRSV-2 ORF1b and Porcine TNF-α.

Porcine reproductive and respiratory syndrome (PRRS) caused by a single-stranded RNA virus (PRRSV) is a highly infectious respiratory disease and leads to huge economic losses to the swine industry worldwide. To investigate the role of miRNAs in the infection and lung injury induced by PRRSV, the differentially expressed miRNAs (DE-miRs) were isolated from PRRSV-2 infected/mock-infected PAMs of Meishan, Landrace, Pietrain, and Qingping pigs at 9, 36, and 60 hpi. Mir-331-3p was the only common DE-miR in each set of miRNA expression profile at 36 hpi. Mir-210 was one of 7 common DE-miRs between PRRSV infected and mock-infected PAMs of Meishan, Pietrain, and Qingping pigs at 60 hpi. Mir-331-3p/mir-210 could target PRRSV-2 ORF1b, bind and downregulate porcine TNF-α/STAT1 expression, and inhibit PRRSV-2 replication, respectively. Furthermore, STAT1 and TNF-α could mediate the transcriptional activation of MCP-1, VCAM-1, and ICAM-1. STAT1 could also upregulate the expression of TNF-α by binding to its promoter region. In vivo, pEGFP-N1-mir-331-3p could significantly reduce viral replication and pathological changes in PRRSV-2 infected piglets. Taken together, Mir-331-3p/mir-210 have significant roles in the infection and lung injury caused by PRRSV-2, and they may be promising therapeutic targets for PRRS and lung injury/inflammation.

An Outbreak of a Respiratory Disorder at a Russian Swine Farm Associated with the Co-Circulation of PRRSV1 and PRRSV2.

We conducted a cross-sectional study to identify the major respiratory pathogen responsible for an outbreak of respiratory disease at a swine farm in West Siberia in 2019. We discovered that the peak of morbidity and mortality coincided with a high level of porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2-related viremia. Based on longer PRRSV2 viremia, the dominant role of PRRSV2 over PRRSV1 in the outbreak was assumed. Phylogenetic analysis revealed that the PRRSV1 strain belonged to sub-genotype 2-one of the predominant groups of genotype 1 PRRSVs in Russia. A partial open reading frame 7 sequence of the PRRSV2 isolate demonstrated a high identity with modified live vaccine-related strains from Denmark (93%) and wild-type VR2332 (92%). We identified the first instance of PRRSV1/PRRSV2 mixed infection in Russia. This finding indicates that further field investigations are needed to access PRRSV2 epidemiology in eastern Europe.

Novel ORF5 deletion of NADC30-like porcine reproductive and respiratory syndrome viruses circulating in northern China from 2016 to 2018.

North American porcine reproductive and respiratory syndrome virus (NA-PRRSV), especially NADC30-like PRRSV, has evolved and is prevalent in China. We collected 503 samples from pig breeding farms across 4 provinces in northern China from 2016 to 2018. The samples were screened by PCR testing with specific primers that could differentiate groups of NA-PRRSV; phylogenetic trees were constructed and analyzed. Overall, 175 of 503 (34.8%) samples were positive for NA-PRRSV. Dual (NADC30-like and highly pathogenic [HP]-PRRSV; NADC30-like and typical PRRSV; HP and typical PRRSV) and triple (NADC30-like, HP, and typical PRRSV) infections (92 of 175, 52.6%) were common in coinfections by NADC30-like and HP-PRRSV. Notably, 18 of 125 (14.4%) semen samples were positive for PRRSV, and 17 of the 18 positive semen samples contained NADC30-like PRRSV. Phylogenetic analysis based on GP5 amino acids revealed that the novel NADC30-like PRRSV with a unique single amino acid deletion at position 34 has become widespread and has evolved into a new subgroup.

Full-genome analysis and pathogenicity of a genetically distinct Russian PRRSV-1 Tyu16 strain.

Porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) strains from Eastern Europe have a high diversity. All three known subtypes (1, 2, 3) of PRRSV-1 have been detected in Russia. There are two different groups of viruses belonging to the subtype 1: pan-European subtype 1 strains, and insufficiently studied Russian strains. The main objective of this study was to characterize the full genomic structure of the atypical Tyu16 strain of the Russian group subtype 1 PRRSV-1 and to assess its pathogenicity. Complete sequencing of the Tyu16 strain revealed that it did not belong to any existing subtype. Comparison of the whole genome sequence of the Tyu16 strain with that of PRRSV-1 prototype strains revealed 78.1 % (subtype 1 Lelystad), 78.1 % (subtype 2 WestSib13) and 77.7 % (subtype 3 Lena) nucleotide identity level, respectively. The coding sequence of different parts of the Tyu16 strain genome demonstrated a varying percentage identity to the different reference PRRSV-1 strains, which may indicate recombination events in its evolutionary history. We assume that among PRRSV-1 isolates, the Tyu16 is the closest relative to the common ancestor of PRRSV-1 and PRRSV-2. Low pathogenicity of the Tyu16 was demonstrated by experimental infection of 70-day-old piglets. Infected animals showed fever not exceeding 7 days, dyspnea in two out of five pigs and reduced weight gain. The virus shedding was undetectable and viremia was at low level.

Novel lineage 1 recombinants of porcine reproductive and respiratory syndrome virus isolated from vaccinated herds: genome sequences and cytokine production profiles.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated, macrophage-tropic arterivirus that exhibits profound genetic and pathogenic heterogeneity. The present study was conducted to determine the complete genome sequences of two novel Korean lineage 1 PRRSV-2 strains, KNU-1901 and KNU-1902, which were isolated from vaccinated pig farms experiencing unusually high morbidity and mortality. Both isolates contained notable discontinuous 423-nucleotide deletions (DELs) within the genes encoding nonstructural protein 2 (nsp2) and GP3 when compared with the prototype strain VR-2332. In particular, the nsp2 DEL viruses had unique quadripartite discontinuous DEL signatures (111-1-19-9) in nsp2; this is an expanded version of the tripartite 111-1-19 DEL previously identified in virulent lineage 1 PRRSV-2 strains. Phylogenetic analysis revealed that both novel nsp2 DEL viruses belong to the Korean clade (KOR C) of lineage 1 isolates based on ORF5 but cluster with lineage KOR A strains based on the nsp2 or complete genome sequence. Recombination detection analysis suggested that both novel isolates are recombinants and may have evolved via natural inter-lineage recombination between circulating KOR A and KOR C strains. Interestingly, compared with the prototype VR-2332 virus, the novel nsp2 DEL variants were less efficient at promoting the expression of immune response genes in porcine alveolar macrophage culture. Taken together, we conclude that KNU-1901 and KNU-1902 are recently evolved recombinant variants of the virulent lineage 1 family that caused the regional severe PRRS outbreaks.

Porcine reproductive and respiratory syndrome virus: phylogenetic analysis of circulating strains in the Republic of Ireland from 2016 to 2017.

In order to investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains currently circulating in the Republic of Ireland (ROI), the ORF5 gene from 17 field strains originating from four vaccinating commercial herds was sequenced and phylogenetically analysed. High genetic variability was observed between farms at the nucleotide (86.3-95.2%) and amino acid (85.5-96%) levels. Phylogenetic analysis confirmed that all field strains belonged to the European species (type 1) and clustered into three separate groups within the subtype 1 subgroup. This variation may pose challenges for diagnosis and prophylactic control of PRRSV through vaccination in the ROI.

Immune response and protective efficacy of intramuscular and intradermal vaccination with porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) modified live vaccine against highly pathogenic PRRSV-2 (HP-PRRSV-2) challenge, either alone or in combination with of PRRSV-1.

The study was conducted to evaluate the immune response of pigs vaccinated intramuscularly (IM) or intradermally (ID) with porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) modified live vaccine (MLV). The protective efficacy was evaluated upon challenge with highly pathogenic (HP)-PRRSV-2, either alone or in combination with PRRSV-1. Forty-two, castrated male, PRRSV-free pigs were randomly allocated into 7 groups of 6 pig each. IM/HPPRRSV2, IM/CoChallenge, ID/HPPRRSV2 and ID/CoChallenge groups were vaccinated IM or ID with PRRSV-1 MLV (UNISTRAIN® PRRS, Laboratorios Hipra S.A., Amer, Spain) in accordance to the manufacturer's directions. NV/HPPRRSV2 and NoVac/CoChallenge groups were nonvaccinated/challenged controls. NoVac/NoChallenge group was left as the control. Antibody response, IFN-γ-secreting cells (IFN-γ-SC) and IL-10 production were evaluated following vaccination. At 35 days post vaccination (DPV), all challenged groups were intranasally inoculated with HP-PRRSV-2, either alone or in combination with PRRSV-1. PRRSV viremia and lung lesion scores were evaluated following challenge. The results demonstrated that ID vaccinated pigs had significantly lower IL-10 levels and higher IFN-γ-SC than that of IM vaccinated pigs. Following challenge with HP-PRRSV-2 either alone or with PRRSV-1, PRRSV viremia and lung lesions, both macroscopically and microscopically, were significantly reduced in vaccinated pigs than that of nonvaccinated pigs, regardless to the route of vaccine administration. ID vaccinated pigs had significantly lower levels of PRRSV viremia and lung lesion scores than that of IM vaccinated pigs. The results of the study suggested that the administration of PRRSV-1 MLV, either IM or ID, provided partial protection against HP-PRRSV-2, either alone or when cochallenged with PRRSV-1, as demonstrated by the reduction in lung lesions and viremia. The ID route might represent an alternative to improve vaccine efficacy, as it resulted in lower IL-10 levels and higher IFN-γ-SC levels.

PCV2 co-infection does not impact PRRSV MLV1 safety but enhances virulence of a PRRSV MLV1-like strain in infected SPF pigs.

Co-infection by a type 1 modified live vaccine-like strain (MLV1-like) of porcine reproductive and respiratory syndrome virus (PRRSV) and a type 2 porcine circovirus (PCV2) was identified on a French pig farm with post-weaning multisystemic wasting syndrome (PMWS). An in vivo experiment was set up to characterize the virulence level of the MLV1-like strain compared with the parental MLV1 strain, and to assess the impact of PCV2 co-infection on the pathogenicity of both PRRSV strains. Six groups of six pigs each were inoculated only with either one of the two PRRSV strains or with PCV2, or co-inoculated with PCV2 and MLV1 or PCV2 and MLV1-like strains. Six contact pigs were added to each inoculated group to assess viral transmission. The animals were monitored daily for 35 days post-inoculation for clinical symptoms. Blood and nasal swabs were sampled twice a week, and tissue samples were collected during necropsy for viral quantification. Compared to MLV1-infected pigs, animals infected with the MLV1-like strain had increased PRRSV viremia and nasal shedding, a higher viral load in the tonsils, and lymph node hypertrophy at microscopic level. PCV2 co-infection did not influence clinical, virologic or transmission parameters for MLV1, but co-infected MLV1-like/PCV2 pigs had the most severe lung lesions, the highest viremia in contact animals and the highest transmission rate. Our study demonstrated that the MLV1 strain tested was safe when co-inoculated with PCV2 in piglets. However, co-infection by the MLV1-like strain and PCV2 resulted in increased virulence compared with that due to a single infection.