Porcine reproductive and respiratory syndrome (PRRS) remains one of the most important infectious diseases affecting the global swine industry. The recent emergence of a highly pathogenic PRRSV-1 strain in northeastern Spain (first detected in 2020) has presented new challenges for disease control. Although these virulent strains are devastating in the field, their comprehensive experimental characterization has yet to be fully investigated. Therefore, this study systematically investigated the genetic origins, in vitro replication characteristics, and pathogenicity mechanisms of the newly emerged, highly pathogenic Spanish PRRSV-1 strain, Lleida 029_22, via dual routes of infection (intramuscular and intranasal).
A recent study published in Wiley Online Library analyzed the genetic characteristics and pathogenicity of the highly pathogenic PRRSV-1 strain Lleida 029_22 in pigs. Phylogenetic analysis revealed that the Lleida 029_22 strain belongs to a new clade associated with the PRRSV-1 Rosalia outbreak and is homologous to the highly pathogenic Italian strain PR40. This strain replicates efficiently in porcine alveolar macrophages and PAM-KNU cells in vitro, but not in MARC-145 cells.
To assess its pathogenicity, eight-week-old piglets were inoculated with the same dose of 2×10⁵ TCID⁵ of Lleida 029_22 via intramuscular (IM) and intranasal (IN) injections. IM inoculation resulted in 100% mortality within 14 days, accompanied by high viremia, high viral shedding, significantly elevated levels of proinflammatory cytokines (particularly IL-6), and severe lung lesions. In contrast, pigs vaccinated with the IN vaccine had a lower mortality rate (30%) and moderate clinical signs. Survivors recovered after 63 days, but exhibited prolonged viremia and shedding, and low levels of proinflammatory cytokines and neutralizing antibodies from day 28 onward.
Interestingly, IN infection faithfully recapitulated the clinical symptoms of an outbreak caused by the highly virulent Rosalia strain on Spanish farms, while IM infection highlighted the risk of nosocomial transmission.
Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen threatening the global swine industry. Its high variability and diversity make existing vaccines inconsistently effective. In 2020, a highly virulent PRRSV-1 strain named "Rosalia" emerged in Spain and spread rapidly, causing severe losses (e.g., increased mortality during the growing season). Genetic analysis suggests that it likely arose from a highly virulent Italian strain, PR40, through recombination. This strain causes high abortion and mortality rates (>20%) in the field, but systematic experimental data are lacking.
Study Objectives and Methods: This study aimed to comprehensively characterize the highly virulent PRRSV-1 strain isolated from the Rosalia outbreak using in vitro and in vivo experiments. Eight-week-old piglets were inoculated via both intramuscular (IM) and intranasal (IN) routes of infection (IM is the commonly used experimental route, while IN simulates natural infection).
Testing Objectives: Detailed documentation of clinical signs and mortality; gross, microscopic, and immunohistochemical pathological examinations of deceased/necropsied pigs; viral loads in serum, saliva, nasal swabs, rectal swabs, and tissues, as well as virus isolation, were performed to understand viral dynamics and transmissibility; and immune responses were assessed by analyzing PRRSV-specific and neutralizing antibody levels, as well as serum cytokine levels.
Key Significance: This study will provide a key experimental model and data foundation for evaluating the efficacy of existing vaccines, developing new vaccines, and further understanding the pathogenesis of this highly virulent strain.
Results
1. Phylogenetic Analysis of PRRSV-1 Strains and Nsp2 Amino Acid Sequence Characterization
Genomic Characteristics and Phylogenetic Positioning:
Deep sequencing (286×) confirmed that the PRRSV-1 Lleida 029_22 strain genome is 14,858 nt long (Figure 1A), phylogenetically belonging to the PRRSV-1 subtype 1 lineage. This strain forms an independent clade from three other Rosalia outbreak-associated strains (Figure 1A), sharing nucleotide identity of 96.61%-97.26%, and is homologous to the highly pathogenic PR40 strain.
Nsp2 Deletion Characterization:
The Nsp2 protein of the Lleida 029_22 strain exhibits a 63-amino acid deletion (positions 317-379 relative to the Lelystad strain) (Figure 1B). This deletion is conserved within the Rosalia clade, while the deletion in its homologous strain, PR40, is even larger (Figure 1A), suggesting that this genetic variation arose early in the evolution of the clade.
Figure 1. Phylogenetic tree of PRRSV-1 Lleida 029_22 strain and Nsp2 sequence alignment
(A) Maximum likelihood phylogenetic tree constructed based on the whole genome sequence (GTR model, 1000 bootstrap tests).
▲: Lleida 029_22 strain used in this study; ●: Rosalia outbreak-associated strain.
(B) Partial Nsp2 amino acid sequence alignment (CLUSTAL Genomics Workbench 24.0.1).
Compared strains: Lleida 029_22, Rosalia outbreak-associated strain, highly virulent PR40 strain, and prototype Lelystad strain.
*Note: Nsp2, nonstructural protein 2; PRRSV-1, porcine reproductive and respiratory syndrome virus type 1.
2. In vitro replication characteristics of PRRSV-1 Lleida 029_22 strain
First, the replication characteristics of the Lleida 029_22 strain were evaluated in vitro. This strain replicated efficiently in PAMs and PAM-KNU cells, but not in MARC-145 cells (Figure 2A). Viral growth curves in the three cell types showed similar viral titers in PAMs and PAM-KNU cells, although the latter had slightly lower titers (Figure 2B). At 72 hours, both cell types reached viral titers of approximately 10⁵TCID₅₀/mL. In contrast, MARC-145 cells were unable to sustain efficient viral infection, with viral titers falling to undetectable levels after 72 hours.
Figure 2. Growth Kinetics of PRRSV-1 Lleida 029_22 in Three Cell Lines
(A) Fluorescence microscopy 72 hours after infection (MOI = 0.1, staining with anti-N protein antibody; 20x objective)
(B) Dynamics of viral titers in PAM-KNU cells (mean ± SD of three independent experiments)
*Note: MOI, multiplicity of infection; PAM, primary alveolar macrophages; PRRSV-1, porcine reproductive and respiratory syndrome virus type 1.
3. Clinical Manifestations and Mortality in Pigs Infected with Lleida 029_22
The pathogenicity of PRRSV-1 Lleida 029_22 was assessed by intramuscular (IM) and intranasal (IN) inoculation routes. Pigs in the IM group showed an increase in body temperature starting at 1 dpi, exceeding 41°C at 7 dpi, and persisting until 13 dpi. The intranasal inoculation group exhibited a milder fever, only briefly exceeding 41°C at 5 days post-inoculation, followed by an intermittent fever pattern. The control group exhibited no abnormal body temperatures throughout the experiment (Figure 3A).
Clinical observations revealed that the IM-infected group experienced acute respiratory distress, neurological symptoms, severe dyspnea, and cyanosis of the ears and scrotum, while the IN-infected group primarily presented with moderate, persistent dyspnea. Additional clinical signs included ragged fur, edema (most pronounced on the limbs and neck), joint inflammation, and overall deterioration in body condition in both groups, but these were more severe in the IM group. These severe clinical manifestations were primarily concentrated between 12 and 14 days post-inoculation, coinciding with the period of high mortality or euthanasia for welfare reasons.
Overall, clinical scores in the IM-infected group were significantly higher than those in the IN-infected group at 11, 12, and 14 days post-inoculation (p < 0.05), peaking at 14 days post-inoculation (Figure 3B). Clinical scores in the IN group were lower, reaching their peak at 13 days post-infection (dpi). Symptoms gradually subsided thereafter, but scores remained significantly higher than those in the control group (p < 0.05). No clinical symptoms were observed in the control group throughout the experiment (Figure 3B).
Linear mixed model analysis (accounting for the non-independence of observations from the same animal at different time points) revealed that group- and day-specific variables significantly influenced body temperature and clinical scores during the first 14 days (p < 0.05). Significant differences were found between the IM and IN groups, as well as between both infection groups and the control group.
Survival analysis showed that all animals in the IM infection group died before 14 days post-infection, with a 100% mortality rate. Natural or induced deaths were concentrated between 12 and 14 days post-infection. In the IN infection group, mortality was 30% by 63 days post-infection, with deaths occurring at 12, 21, and 43 days post-infection. All animals in the control group survived until the end of the experiment (Figure 3C).
Figure 3. Clinical Outcomes in Pigs Infected with PRRSV-1 Lleida 029_22
(A) Daily Rectal Temperatures Post-Infection (Red Dashed Line: Fever Threshold >41°C; Mean ± SD)
(B) Daily Mean Clinical Score (Mean ± SD; Identical Letters Indicate No Significant Differences Between Groups, p < 0.05)
(C) Survival Curves for Experimental Groups
Note: C, control group; IM, intramuscular group; IN, intranasal group.
4. Viremia and Viral Shedding in PRRSV-Infected Animals
Viraemia levels following PRRSV-1 Lleida 029_22 infection were assessed by RT-qPCR. PRRSV RNA was detected in the serum of all infected animals at 3 days post-infection, with significant differences between the IM and IN groups compared to the control group, which remained negative (p < 0.05; Figure 4A). Viraemia in the IN group peaked at 7 days post-infection, while viral loads in the IM group were significantly higher than those in the IN and control groups at both 7 and 14 days post-infection (p < 0.05). Thereafter, the viral load in the IN group gradually decreased, but was still significantly higher than that in the control group at 28 dpi.
Figure 4. Viral shedding was assessed by saliva and nasal/rectal swabs. All infected animals tested positive for virus at 3 dpi in saliva, nasal, and rectal swabs.
Salivary shedding continued until 14 dpi in the IM group and until the end of the experiment in the IN group (peak at 3 dpi; Figure 4B).
Nasal shedding peaked at 7 dpi in both groups, with significantly higher viral loads in the IM group at 7/14 dpi than in the IN group (*p<0.05; Figure 4C).
Rectal shedding continued until 14 dpi in the IM group and until 35 dpi in the IN group (with a slight rebound at 56 dpi), with both peaks at 7 dpi (Figure 4D).
AUC analysis confirmed that viral loads in all samples from the infected groups were significantly higher than those in the control group, and that nasal swab loads in the IM group were higher than those in the IN group (*p<0.05). In summary, PRRSV shedding occurs early and is sustained after infection.
5. Verification of Isolation of Infectious PRRSV from RT-qPCR-Positive Samples
To confirm the presence of infectious virus in RT-qPCR-positive samples, virus isolation (VI) experiments were performed using PAM-KNU cells (Figure 5). For serum samples, the success rate of virus isolation was 100% (10/10) for samples collected between 3 and 14 days post-inoculation (dpi) in the IM group and 100% (10/10) for samples collected between 3 and 7 days post-inoculation (dpi) in the IN group. However, the success rate gradually decreased at 14 days post-inoculation (70%), 21 days post-inoculation (55.6%), and 28 days post-inoculation (12.5%).
Figure 5. Three infectious virus strains were successfully isolated from saliva samples collected from both infected animals at 3 days post-infection (dpi) after two passages of culture. No infectious virus was isolated from nasal or rectal swabs.
6. Cytokine Responses Induced by PRRSV-1 Lleida 029_22 Infection
Serum samples were collected during the first 14 days post-infection to assess cytokine levels, revealing significant differences among the three groups (Figure 6). IFN-α levels were significantly elevated in both infection groups at 3, 7, and 14 days post-infection (dpi), with IFN-α levels in the IM group significantly higher than in the IN group at 14 days post-infection (p<0.05). Proinflammatory cytokines IL-1α, IL-12, and IL-6 showed similar trends: IL-1α increased in the IN group at 3 days post-infection and remained stable, while IL-6 levels in the IM group were significantly higher than in the control group at 7 and 14 days post-infection (Figure 6B, D-E). Of particular note, IL-6 levels were significantly higher in the IM group than in the IN group at 14 days post-infection (p<0.05). IL-1β in both groups of infected animals showed an increasing trend, with the IM group showing significantly higher levels than the control group at 3, 7, and 14 dpi, while the IN group showed a significant difference only at 14 dpi ( Figure 6C ).
Figure 6. IL-12 levels were significantly higher in the IM group than in the IN group and control group at 14 dpi (Figure 6F). The overall trend showed a stronger pro-inflammatory cytokine response in the IM group, particularly in IL-6 levels at 7 and 14 dpi, which was positively correlated with the 100% mortality rate in the IM group.
7. Specific and Neutralizing Antibody Responses in Animals Infected with PRRSV-1 Lleida 029_22
PRRSV-1 and PRRSV-2-specific antibodies were assessed using commercial ELISA kits (Figure 7A). Seroconversion occurred in 70% of animals in the IM group and in 90% of animals in the IN group at 14 dpi. The S/P ratio in both groups was significantly higher than that in the control group (p < 0.05). Antibody levels in the IN group began to decline at 21 dpi, remained stable at 42 dpi, rebounded at 56 dpi, and then declined slightly at 63 dpi. The control group remained seronegative throughout the experiment (S/P < 0.4).
Figure 7. Neutralizing antibody testing showed that in the IN group, only a low titer of 2 log₂ was detected at 28 dpi, which then gradually increased to 3.71 log₂ at 56 dpi (Figure 7B). Antibody titers varied among individuals at the same time point, and the appearance of neutralizing antibodies coincided with the clearance of infectious virus from the serum after 28 dpi (Figure 4B). No neutralizing antibodies were detected in the control group.
8. Detection and Isolation of PRRSV-1 Lleida 029_22 in Tissue Homogenates
RT-qPCR was used to measure viral loads in various tissues (Figure 4F). In the IM group, viral loads were relatively consistent across tissues, with the highest loads in the lungs, mediastinal lymph nodes, and spleen. In the IN group, viral loads varied significantly across tissues, with the highest loads in the tonsils and mediastinal lymph nodes. As expected, the overall viral load in the IN group was lower than that in the IM group, which may be related to the different time of necropsy. The significant variability in tissue loads in infected animals in the IN group was also associated with different times of death (grayed-out samples at 12, 21, and 43 dpi versus 63 dpi). No viral RNA was detected in any tissue sample from the control group.
Table 1. Tissues collected from animals that died at 12, 21, or 43 days post-infection tested positive for aVI.
(Table Note: Table 1 summarizes the results of RT-qPCR and virus isolation from tissue samples. "MLN" denotes mediastinal lymph nodes, and "ILN" denotes inguinal lymph nodes.)
9. Pathological and Immunohistochemical Analysis of PRRSV-Infected Tissues
Necropsy findings: Animals that died after PRRSV infection (particularly those that died between 12 and 21 days post-infection) showed significant systemic lesions, including purpuric skin, generalized edema, severe lymphadenopathy, splenomegaly (sometimes with follicular hyperplasia), and gastric hemorrhage. Pulmonary lesions were prominent, demonstrating partial collapse, multifocal consolidation, and reddish-brown macules, consistent with interstitial pneumonia, with more severe lesions in animals that died early (Figure 8A). Approximately 20% of animals with intranasal infection also developed abscesses, pericarditis, and arthritis caused by secondary bacterial infection. No gross lesions were observed in control animals.
Figure 8. Histopathological analysis showed that interstitial pneumonia scores in animals that died early (12-21 dpi) in the IM and IN groups ranged from 1.83 to 3.33 points. However, these scores were significantly reduced in animals sacrificed at 63 dpi (control group, 0.17-0.83 points; IN group, 0.50-1.17 points). Of particular note, the tonsil histological score in the IN group was significantly higher than that in the control group at 63 dpi (p<0.05), but no significant differences were observed in other tissues (Figure 8C).
(Caption: Figure 8B shows the histopathological scores, and Figure 8C compares the histological scores of the various groups at 63 dpi)
Microscopic pathology revealed interstitial pneumonia (alveolar wall thickening, cellular proliferation, and inflammatory cell infiltration) in the lungs of all infected animals. Exudates and necrotic tissue were present within the alveoli, along with signs of vascular damage. Lymphoid tissue (lymph nodes and spleen) showed severe lymphopenia and necrosis, accompanied by vascular damage (e.g., thrombosis and vasculitis). Perivascular inflammatory cell infiltration, edema, and gliosis were observed in the brain white matter (Figure 9A).
Immunohistochemical (IHC) analysis detected PRRSV antigens in macrophages, and occasionally in type II pneumocytes, from the lungs, lymph nodes, tonsils, and spleens of infected animals (Figure 9B). Viral antigens were also detected in perivascular inflammatory cells in the brain.
Figure 9. PRRSV infection causes extensive pathological damage in the lungs, lymphoid tissues, and brain, primarily localized within macrophages, exhibiting systemic pathological features.
Discussion
PRRSV has a restricted tropism for monocytic cell lines and has traditionally been isolated and cultured using alveolar macrophages (PAMs) and MARC-145 cells. However, PAMs are difficult to obtain and have large batch-to-batch variability, and highly pathogenic strains are difficult to adapt to MARC-145. In this study, we used the immortalized cell line PAM-KNU, which is sensitive to certain field strains, instead of PAMs. We successfully isolated the Lleida 029_22 strain, whose growth curve was similar to that observed in PAMs. Currently, there is no universal cell line suitable for all PRRSV strains, and future efforts are needed to identify more suitable cell lines for the isolation of diverse strains.
Conclusion
This study successfully established a model of intranasal infection with a highly virulent PRRSV-1 strain. The clinical manifestations (high mortality, severe symptoms, persistent high fever, and viremia) closely resemble those observed in field outbreaks. The key finding was that the route of infection significantly affected the outcome: intramuscular (IM) infection resulted in 100% acute mortality, while intranasal (IN) infection had a mortality rate of only 30%, and the infected pigs eventually recovered completely. This significant difference highlights the risk that on-farm IM injection practices could significantly exacerbate the severity of the epidemic through nosocomial transmission.
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