In 2006, HP-PRRSV, a strain derived from the classical PRRSV, caused an epidemic in China characterized by high fever, morbidity, and mortality. Subsequently, the strain spread widely throughout China and Asia, undergoing significant mutations. HP-PRRSV and its variants have become the dominant strains in China, but research on the epidemiology, molecular evolution, and pathogenicity of the novel L8.7 PRRSV remains limited.
On May 22, 2025, a study titled "Genetic Evolution and Pathogenic Variation of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus" published in Taylor & Francis systematically elucidated the epidemiological dynamics, evolutionary trends, vaccine strain associations, and pathogenicity evolution of the L8.7 lineage.
This study provides key data support for developing L8.7 PRRSV prevention and control strategies.
Abstract
Based on analysis of 2,509 global L8.7 ORF5 gene sequences, the L8.7 lineage was divided into seven groups (L8.7.1-L8.7.7). L8.7.1-L8.7.3 correspond to reported classical PRRSV, intermediate strains, and HP-PRRSV, respectively, while L8.7.4-L8.7.7 are defined as HP-like PRRSVs.
Statistical analysis showed that HP-like PRRSVs predominated within the L8.7 lineage, with L8.7.5 and L8.7.6 strains representing the highest proportion in recent years. Comprehensive whole-genome analysis revealed that 72.15% of L8.7 strains exhibited wild-type characteristics.
Evolutionary rate analysis revealed that the evolutionary rate of the L8.7.3-L8.7.7 lineages in China has decreased approximately 4.1-fold since the introduction of the attenuated HP-PRRSV vaccine (MLV).
Pathogenicity testing revealed that, compared to HP-PRRSV (L8.7.3: HuN4), HP-PRRSV-like strains (L8.7.5: DLF; L8.7.6: DLW) maintain high virulence while exhibiting reduced pathogenicity in piglets.
# Graphical Abstract
Experimental Results
# Classification of L8.7 PRRSV
To analyze the evolutionary characteristics of PRRSV L8.7, this study analyzed a total of 2509 ORF5 sequences: 2159 L8.7 strain sequences were obtained from the NCBI database, and 350 sequences were collected in our laboratory between 2014 and 2023 (Figure 1(a)). As shown in the figure, L8.7 strains were further divided into seven groups (L8.7.1–8.7.7) (Figures 1(a, b)); all sequence information is available (Figure 2).
As shown in Figure 1(b), the reference strains used to construct the phylogenetic tree were from known classical strains and L8.7 PRRSV strains reported in pathogenicity studies. The average genetic distances within and between groups are shown in Figure 1(c). With the exception of L8.7.2, the average genetic distances within all groups were less than 5%. Overall, the genetic distances between groups ranged from 4.3% to 10.4%. In addition, the L8.7.4-L.7.7 strains exhibited specific amino acid mutation patterns with different characteristics and were defined as HP-PRRSV-like. Among the 2509 sequences in the L8.7 population, 2.23% (56/2509) belonged to L8.7.1 (CH-1a-like PRRSV), 4.74% (119/2509) to L8.7.2 (intermediate PRRSV), 11.48% (288/2509) to L8.7.3 (HP-PRRSV), and 81.54% (2046/2509) to HP-PRRSV-like.
Figure 1 Phylogenetic tree and nucleotide identity analysis of L8.7 strains
(a) Phylogenetic tree dividing L8.7 sequences into seven groups. (b) Phylogenetic tree constructed based on the ORF5 gene of L8.7 PRRSV isolates and reference PRRSV strains from each lineage. The experimental strains used in this study are marked with yellow stars. (c) Genetic distances within and between L8.7 strain groups (percentage of nucleotide differences).
Figure 2 Comparative Analysis of Pathogenicity of PRRSV L8.7.1-L8.7.7
# Global Distribution of PRRSV L8.7
This study comprehensively analyzed L8.7 sequences for which temporal and geographic information is known. Notably, group L8.7.4 was the most widespread, covering eight of the nine countries where L8.7 strains were found (Figure 3(a, b)). Nepal, Laos, and Myanmar detected only a single group, L8.7.4; no other groups were found. L8.7.1, 8.7.3, 8.7.5, 8.7.6, and 8.7.7 strains were found in two, three, four, four, and two countries, respectively (Figure 3(a, b)). The L8.7.2 strain has been reported only in China (Figure 3(b)). The number (2201/2509, 87.7%) and diversity (7/7 groups, 100%) of L8.7 PRRSV strains in China ranked first ( Figure 3(b) ).
Figure 3 (a) Geographical distribution of L8.7 strains in different regions of the world. Figure 3 (b) National distribution of L8.7 strains.
This study analyzed a total of 2,201 L8.7 PRRSV strains from China. L8.7 PRRSV infection has been reported in 26 provinces in China, with Guangdong Province reporting the most cases, followed by Guangxi, Heilongjiang, Shandong, Hebei, and Henan, each with over 40 cases reported (Figure 3 (c, d)). The prevalence of different PRRSV groups in China exhibits temporal dynamics (Figure 3 (e)), with distinct peaks in prevalence in specific groups. Groups L8.7.1 and L8.7.2 have been detected at very low rates and have rarely been reported since 2006. Group L8.7.3, after causing an outbreak in 2006, became dominant and persisted from 2006 to 2009.
Figure 3(c) Geographical distribution of L8.7 strains in different regions of China. Figure 3(d) Population distribution of L8.7 strains in various provinces of China.
HP-like PRRSVs (L8.7.4-8.7.7) have gradually replaced HP-PRRSV as the predominant circulating strains in China (Figure 3(e)). Group L8.7.4 was first reported and monitored in China in 2006 and comprised a significant proportion of L8.7 strains in China between 2009 and 2011 (41.3%-79.6%). Notably, some groups have experienced sudden increases in prevalence: for example, group L8.7.5, which first appeared in China in 2007 and has been circulating continuously, has seen a significant increase in prevalence since 2011 (17.1%-51.6%) (Figure 3(f)). The cyclical nature of the L8.7.6 strain is also noteworthy. This group (EU709835.1, SH02) was first identified in 2002. Initially, its detection rate was extremely low (only one strain), and it was not detected between 2003 and 2005. After a rapid increase in 2006, its prevalence gradually decreased until 2009. It then became the predominant strain between 2014 and 2023, accounting for 21.5% to 47.1%. Group L8.7.6 was most frequently detected in China (612/2201, 27.8%) and had the widest distribution (20/21 provinces, 95.2%) (Figure 3(d, f)). Group L8.7.7 was first detected in 2008, but its prevalence remained low until 2011, after which it gradually increased. Its detection rate rapidly increased to 15.1% to 17.1% between 2022 and 2023.
Figure 3 (e) Distribution of L8.7 strains over time based on ORF5 sequences. Figure 3 (f) Stacked bar chart of relative frequencies across China.
These results reveal that over the past decade, L8.7.5 and L8.7.6 strains were not only the most abundant but also the most widely distributed in China.
# Relationship between HP-PRRSV MLVs and HP-PRRSVs
To investigate the association between HP-PRRSV attenuated vaccines (JXA1-R, HuN4-F112, TJM-F92, GDr180) and HP-PRRSV-like strains, this study comprehensively analyzed strains of the L8.7.4–8.7.7 lineages based on nucleotide identity, NSP2 deletion signatures, and genome-wide characteristic amino acid changes (Table 1).
Table 1 Genome-wide association analysis between HP-PRRSV attenuated vaccine (MLV) and HP-PRRSV-like strains
The results indicate that the key to distinguishing vaccine-like PRRSV from HP-PRRSV-like strains cannot be determined by whole-genome nucleotide identity or characteristic amino acid changes, but rather by the presence of additional NSP2 deletions (Table 1). Statistical analysis revealed that 27.85% (22/79) of the L8.7.6 lineage strains were associated with the vaccine.
Pathogenicity of HP-PRRSV and HP-PRRSV-like strains in piglets
# Isolation and identification of dominant HP-PRRSV-like strains
To systematically elucidate the pathogenicity of the dominant HP-PRRSV-like strains (L8.7.5 and L8.7.6), this study successfully isolated the L8.7.5 lineage strain DLF and the L8.7.6 lineage strain DLW. These viruses were isolated from porcine alveolar macrophages (PAMs) and Marc-145 cells. IFA assay showed that PRRSV M protein expression was observed in PAMs and Marc-145 cells inoculated with the strain ( Figure 4(a) ), indicating that the DLF and DLW strains were successfully separated.
Figure 4 Isolation, culture, recombination analysis, and characteristic NSP2 amino acid alignment of the L8.7 strain
(a) Identification of the DLW and DLF strains. Immunofluorescence assay (IFA) using a monoclonal antibody targeting the PRRSV M protein revealed specific reactivity in PAMs and Marc-145 cells from the control, DLF-infected, DLW-infected, and HuN4-infected groups. Cell nuclei were counterstained with DAPI. Scale bar = 300 μm. (b) Analysis of recombination events in DLW. (c) Alignment of the deduced amino acid sequences of the NSP2 proteins of the L8.7 strains.
# Genomic characteristics of DLF and DLW
The full genome lengths of DLF (PQ178809) and DLW (PQ178810) are 15,324 and 15,323 nucleotides, respectively (excluding the poly(A) tail). The genomic nucleotide similarities between HuN4/DLF, HuN4/DLW and DLF/DLW were 98.67%, 95.78% and 95.13%, respectively (as shown in the table below).
Sequence alignment of the NSP2 protein revealed that DLF and DLW exhibited discontinuous deletions of 30 amino acids (1 + 29 amino acids) at positions 482 and 533-561 in the CH-1a strain NSP2 protein. This deletion pattern is consistent with that of L8.7.3 (HP-PRRSV) (Figure 4(c)). To investigate whether DLF and DLW were involved in a recombination event, analysis using SimPlot and RDP4 software revealed that DLW experienced a recombination event (recombination sites spanning nt 3500-5657), while DLW did not (Figure 4(b)). Based on both previous studies and the criteria used in this study to distinguish vaccine strains from wild-type strains, both DLF and DLW were wild-type strains.
# Clinical Signs of Infected Piglets
Challenged piglets were weighed every 7 days, and blood samples were collected at 0, 3, 5, 7, 10, 14, and 21 days per iod. The experimental procedure is shown in Figure 5(a).
Figure 5(a) Experimental Design
Piglets in the HuN4 and DLF challenge groups both developed obvious clinical symptoms (coughing, lethargy, indigestion, and chills) by 2 days post-exposure. Piglets in the DLW challenge group displayed typical clinical symptoms of PRRSV infection by 3 days post-exposure, with 3 of 5 infected pigs experiencing coughing, lethargy, indigestion, and shivering. Piglets in the HuN4 challenge group maintained a high fever (≥40.5°C) for 4–6 days (Figure 5(b)) and began to die by 12 days post-exposure. The survival rate was 20% by 21 days post-exposure (Figure 5(c)). Piglets in the DLF challenge group began to die by 16 days post-exposure, with a survival rate of 60% by 21 days post-exposure (Figure 5(c)). Despite a lower mortality rate, the duration of fever in this group was longer (7–15 days) (Figure 5(b)). Piglets in the DLW challenge group survived until the end of the experiment, with a shorter duration of fever (1–8 days) (Figure 5(b)). The piglets in the control group did not show any obvious clinical symptoms and survived throughout the study (Figure 5(b, c)).
Figure 5(b) Rectal temperature trends after challenge with DLF, DLW, and HuN4.
Figure 5(c) Survival rates after challenge with DLF, DLW, and HuN4.
Piglet weights were measured at 0, 7, 14, and 21 dpi. Statistical analysis showed that the average daily weight gain (ADG) of piglets in the DLF-challenged group was significantly lower than that of uninfected piglets from 1 to 7 dpi, 8 to 14 dpi, and 15 to 21 dpi (Figure 5(d)). Compared with uninfected piglets, the ADG of piglets in the HuN4-challenged group was significantly lower from 8 to 14 dpi, while the ADG of piglets in the DLW-challenged group was significantly lower from 8 to 14 dpi and from 15 to 21 dpi (Figure 5(d)).
Figure 5(d) Average Daily Weight Gain Changes in DLF, DLW, and HuN4-Challenged Groups
Data are presented as mean ± standard deviation (error bars). :p<0.05; :p<0.01; :p<0.001; ****:p<0.0001; ns: not statistically significant.
# Dynamic Changes in PRRSV-Specific Antibodies
Blood samples were collected from all pigs at 0, 3, 5, 7, 10, 14, and 21 dpi, and antibodies specific to the PRRSV N protein were detected using a commercial ELISA kit. Results showed that PRRSV-specific antibodies (S/P ratio ≥ 0.4) were detected in piglets in the DLF and HuN4-challenged groups at 10 dpi. By 14 dpi, all five piglets in the DLW-challenged group were antibody-positive (S/P ratio ≥ 0.4). The S/P ratios in the challenged groups continued to increase until the end of the experiment, while no PRRSV-specific antibodies were detected in the unchallenged group throughout the experiment (Figure 5(e)).
Figure 5(e) Changes in anti-PRRSV antibody titers induced by challenge with DLF, DLW, and HuN4.
# Assessment of Viremia and Viral Load in Different Tissues
RT-qPCR was used to analyze viral load distribution in serum samples and 10 tissues obtained at autopsy at 0, 3, 5, 7, 10, 14, and 21 days post-challenge. Results showed that viremia levels in the challenged groups began to increase starting at 3 days post-challenge, peaking at 5 days post-challenge in the DLF and DLW groups and at 7 days post-challenge in the HuN4 group (Figure 5(f)). Viral loads then gradually declined. Significant differences in viremia levels were observed between 7 and 10 days post-challenge (Figure 5(f)). No viremia was detected in the control group throughout the entire challenge period. Although differences in viral loads in the same tissues were observed among the challenged groups, these differences were not statistically significant (Figure 5(g)). ORF7 gene sequencing confirmed that the samples contained the original challenge strain.
Figure 5(f) Dynamic changes in viremia induced by DLF, DLW, and HuN4 challenge
Figure 5(g) Analysis of viral load in various tissues of the DLF, DLW, and HuN4 challenge groups
# Gross and Histopathological Lesions
All HuN4-infected piglets showed severe thymic atrophy (Figure 6(a)). Four piglets in the DLF-challenged group exhibited significant thymic atrophy, whereas no thymic atrophy was observed in the DLW-challenged group (Figures 6(b,c)).
All five pigs in the HuN4-challenged group exhibited lung consolidation (Figure 6(e)), of which four had mandibular lymph node hemorrhage (Figure 6(m)). Of the five pigs in the DLF-challenged group, three had lung consolidation (Figure 6(f)) and three had mandibular lymph node hemorrhage (Figure 6(n)). Two of the five pigs in the DLW-challenged group exhibited lung consolidation (Figure 6(g)), and two pigs had mild hemorrhage in the mandibular lymph nodes (Figure 6(o)). No significant pathological changes were observed in the organ tissues of the uninfected piglets (Figure 6(d,h,p)).
Pigs challenged with HuN4 developed severe interstitial pneumonia with hemorrhage (Figure 6(i)), characterized by thickening of the alveolar septa and mononuclear cell infiltration. Microscopic lesions in the lungs of the DLF and DLW challenged groups were similar, but differed in severity (Figure 6(j,k)). The DLF challenged group showed extensive inflammatory cell infiltration with serous exudates, necrosis and exfoliation of alveolar epithelial cells, and significant necrosis and exfoliation of bronchial epithelial cells (Figure 6(j)). The DLW challenged group showed extensive inflammatory cell infiltration and moderate widening of the alveolar septa (Figure 6(k)). Furthermore, compared with the control group, the challenged groups showed varying degrees of medullary hemorrhage in the mandibular lymph nodes (Figure 6(q-t)), whereas no pathological lesions were observed in these tissues in the control pigs (Figure 6(i,t)).
Figure 6 Gross and Histological Lung Lesions in Different Challenge Groups
Piglets challenged with HuN4 or DLF displayed varying degrees of thymic atrophy (a, b). Compared with the control group, the HuN4 and DLF infection groups developed severe interstitial pneumonia with pulmonary consolidation (e, f) and lymph node hemorrhage (i, j), while the DLW infection group exhibited milder interstitial pneumonia with pulmonary consolidation (g) and lymph node hemorrhage (o). Lung tissues from the challenged groups displayed varying degrees of interstitial pneumonia, characterized by extensive inflammatory cell infiltration, alveolar epithelial hyperplasia, and widening of the alveolar septa (i-k). Furthermore, medullary hemorrhages were observed in the mandibular lymph nodes of the challenged groups (q-t), but not in the control group (r).
Conclusion
The L8.7 lineage is the earliest PRRSV lineage discovered in China and has been circulating for over 25 years. In 2006, HP-PRRSV, a strain derived from classical PRRSV, caused an epidemic in China characterized by high fever, morbidity, and mortality. Subsequently, this strain spread widely throughout China and Asia, undergoing significant mutation. Notably, HP-PRRSV and its variants have become the dominant strains in China, but research on the epidemiological patterns, molecular evolution, and pathogenicity of the novel L8.7 PRRSV remains underdeveloped. Therefore, this study focused on L8.7 PRRSV and conducted a comprehensive and systematic analysis.
The focus of L8.7 strains has primarily been on their pathogenicity, particularly since the 2006 outbreak caused by the L8.7.3 strain (HP-PRRSV). Therefore, this study isolated and evaluated the pathogenicity of the most dominant HP-PRRSV-like strains (L8.7.5: DLF; L8.7.6: DLW) within the L8.7 lineage. Results showed that although the virulence of HP-like PRRSV (DLF and DLW) was reduced compared to HP-PRRSV (HuN4) (as evidenced by increased piglet survival, increased daily weight gain, decreased fever temperature and duration, and differences in thymic atrophy), it still maintained significant pathogenicity. Virulence in this study correlated with serum viral load in challenged piglets at 7 and 10 days per 1000 (dpi), while no significant differences were observed at other time points. Therefore, survival rate, fever temperature and duration, and thymic atrophy are important indicators of PRRSV pathogenicity in piglets.
Since the L1 lineage PRRSV became prevalent in China in 2016, reports of recombinant strains have increased. The predominant recombination patterns in China are L1 (L1.5 or L1.8) with L8.7 or L8.7 with L1 (L1.5 or L1.8). In this study, DLW was a recombinant strain of L8.7 and L1.8 (recombination pattern: L8.7+L1.8), and its pathogenicity was lower than that of HuN4 and DLF. Although DLW showed significantly reduced pathogenicity in piglets, the relationship between the reduced virulence and the recombination event requires further investigation. Overall, with the exception of strains L8.7.1 (low pathogenicity) and L8.7.2 (low pathogenicity), strain L8.7.3 was highly pathogenic in piglets. Combined with previously reported pathogenicity experiments on strains L8.7.5 (2006) and L8.7.6 (2017), HP-like PRRSVs (L8.7.4–8.7.7) maintained high virulence while exhibiting reduced pathogenicity in piglets compared to strain L8.7.3 (see Figure 2).
Figure 2 Comparative Analysis of the Pathogenicity of PRRSV L8.7.1-L8.7.7
# Discussion Viewpoints
1. Clinical Implications of Pathogenicity Evolution
Animal experiments have shown that HP-like strains (such as DLW/L8.7.6), while having a lower mortality rate than classic HP-PRRSV (HuN4), can cause persistent high fever (>41°C), growth inhibition, and elevated lymph node viral loads. This explains why some cases, despite not experiencing acute death, develop persistent respiratory symptoms and secondary infections. It is recommended that ORF5 sequencing and histopathological analysis be combined during diagnosis to avoid misclassifying HP-like strains as low-pathogenic strains.
2. Optimization of Prevention and Control Strategies
Given the "anti-gravity effect" of viral transmission (biosafety measures in large-scale pig farms reduce infection risk), small and medium-sized farms should be the focus of prevention and control. Furthermore, the rapid spread of the L8.7.6 strain within pig herds requires upgraded closed-loop management and enhanced testing.
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