The porcine reproductive and respiratory syndrome virus (PRRSV), especially the highly pathogenic strains, can cause serious acute lung injury (ALI), characterized by extensive hemorrhage, inflammatory cells and serous fluid infiltration in the lung vascular system. Meanwhile, the pulmonary microvascular endothelial cells (PMVECs) are essential for forming the air–blood barrier and keeping the water–salt balance to prevent leakage of circulating nutrients, solutes, and fluid into the underlying tissues. As well, they tightly regulate the influx of immune cells. To determine the possible relationship between the PMVECs’ function changes and lung vascular permeability during PRRSV infection, the PMVECs were co-cultured with HP-PRRSV-inoculated primary pulmonary alveolar macrophages (PAMs) in transwell model, and then the RNA sequencing (RNA-seq) and comprehensive bioinformatics analysis were carried out to characterize the dynamic transcriptome landscapes of PMVECs. In total, 16,489 annotated genes were identified, with 275 upregulated and 270 downregulated differentially expressed genes (DEGs) were characterized at both 18 and 24 h post PRRSV inoculation. The GO terms and KEGG pathways analysis indicated that the immune response, metabolic pathways, cell death, cytokine–cytokine receptor interaction, viral responses, and apoptotic process are significantly regulated upon co-culture with PRRSV-infected PAMs. Moreover, according to the TERR and dextran flux assay results, dysregulation of TJ proteins, including CLDN1, CLDN4, CLDN8, and OCLN, is further confirmed to correlate with the increased permeability of PMVECs. These transcriptome profiles and DEGs will provide valuable clues for further exploring the roles of PMVECs in PRRSV-induced ALI in the future.

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.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen for the global pork industry. Modified live virus (MLV) vaccines are widely used for PRRSV prevention and control. However, MLV vaccines can still infect hosts and replicate in the target cells, increasing the risk of virus recombination and reversion to virulence. Recently, a highly pathogenic PRRSV strain appeared in December 2024, causing severe outbreaks on a PRRSV-negative pig farm in Yunnan, China. The isolated strain exhibited 99.4%–99.5% nucleotide similarity to the historical JXA1-R vaccine-derived revertant isolated in 2012, despite the farm never using JXA1-R vaccines. Ten unique amino acid mutations confirmed that this strain was a revertant derived from JXA1-R vaccines, which reverted to cause an outbreak in 2012 and vanished for 12 years. IMPORTANCE: Currently, there is a relative consensus that porcine reproductive and respiratory syndrome modified live virus (PRRS MLV) has the highest protective efficacy against the genetically homologous virus, compared with commercially available killed vaccines or other kinds of vaccines under development. However, risks of virulence reversion remain in such live vaccines. This case reveals the unpredictable risks of live-attenuated vaccines. The findings underscore the inherent flaws in highly pathogenic PRRSV MLV vaccines, emphasizing the need for safer vaccine designs. The re-emergence of that revertant draws our attention to its source, and the similar mutation site compared with 12 years ago could indicate key nucleotide sites for the reversion of virulence of this strain.

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the global swine industry. Gilt immunization using modified live virus (MLV) vaccines is crucial for herd stability, but it is complicated by frequent mixed infections of PRRSV strains on farm. This study monitored the administration of tylvalosin during a PRRSV-2 MLV (TJM) immunization program, focusing on viral dynamics and immune responses in gilts naturally exposed to co-circulating classical (GD240101) and highly pathogenic like (HP-PRRSV-like, GD240102) PRRSV strains. Methods: The animal study was approved by the Laboratory Animal Ethical Committee of China Agricultural University. One hundred gilts were randomized into control and tylvalosin groups (n = 50/group). All received the TJM MLV vaccination. The tylvalosin group received tylvalosin tartrate premix cyclically in-feed for three cycles. Serum and saliva samples were collected periodically. PRRSV RNA (RT-qPCR) and specific antibodies (ELISA) were assessed. Viral population dynamics (relative abundance, mutation, recombination of TJM, GD240101, and GD240102) were monitored via next-generation sequencing (NGS) on a pooled PRRSV-positive sample. Results: In this field trial where tylvalosin was used, a shorter duration of PRRSV viremia and saliva shedding was observed to compare with controls. NGS analysis showed accelerated vaccine strain (TJM) clearance in the tylvalosin group (by week 3 vs. week 9 in control). Field strain dynamics were also altered, showing a faster decline in the tylvalosin group. Antibody response uniformity was altered, with lower coefficient of variation (CV) for PRRSV and CSFV observed following tylvalosin usage. Conclusions: In gilts receiving tylvalosin for the management of bacterial pathogens during a PRRSV MLV immunization program, it was associated with accelerated viral clearance and enhanced systemic immune response uniformity under mixed-infection field conditions. NGS provides invaluable data for dissecting these complex viral dynamics. Crucially, these findings describe a biological drug–host–virus interaction and should not be interpreted as an endorsement for the prophylactic use of antimicrobials. In alignment with global antimicrobial stewardship principles, tylvalosin should be reserved for the therapeutic treatment of diagnosed bacterial diseases to mitigate the risk of promoting resistance.

Recombination is a primary driver of porcine reproductive and respiratory syndrome virus (PRRSV) evolution. Currently, lineage 1 NADC30-like virus (L1C) and lineage 8 HP-PRRSV (L8E) recombinants dominate in China, whereas recombinants harboring the lineage five backbone are infrequently detected. To elucidate the mechanisms underlying this disparity, we investigated the interplay between recombination frequency, viral fitness, and strain-specific phenotypes using co-infection models. Next-generation sequencing (NGS) revealed that the lineage 5 replicase is prone to template switching, generating high frequencies of recombination junctions comparable to NADC30-like strains when viral RNA abundance is high. However, plaque purification demonstrated a disconnection between the generation of recombination events and the survival of viable progeny. While NADC30-like strains acted as a highly recombinogenic backbone, yielding up to 100% recombinant progeny, lineage 5-based recombinants were largely eliminated due to competitive exclusion. Furthermore, although a mutation known to reduce recombination frequency (K541R) successfully reduced errors at the nucleic acid level, it failed to prevent the emergence of recombinants under strong selective pressure. Our findings suggest that PRRSV recombination is not merely a stochastic event but a complex outcome dictated by strain-specific phenotypic profiles. The scarcity of lineage 5 recombinants is driven by a phenotypic deficit, which is a combination of replication efficiency and susceptibility to host clearance. This study highlights the dominant role of NADC30-like strains in driving viral diversity and underscores that vaccine safety depends on the holistic phenotypic fitness of the virus. IMPORTANCE: Porcine reproductive and respiratory syndrome virus (PRRSV) recombination poses a significant threat to the farm as it accelerates viral evolution and leads to changes in pathogenicity and immune protection. Understanding why certain strains recombine frequently while others do not is critical for reducing this risk. This study unravels the complexity of PRRSV recombination, showing that it is determined by the specific phenotypic profile of the virus strains involved. We discovered that current epidemic strains (NADC30-like) act as highly recombinogenic backbones due to their biological traits. Surprisingly, classical vaccine strains (lineage 5) infrequently form viable recombinants not because they are genetically accurate, but because their distinct phenotypic weaknesses lead to their elimination by the host or competitors. This finding shifts the paradigm of vaccine safety, emphasizing that preventing recombination requires a holistic approach that considers the complex interplay of viral fitness and host adaptation, thereby providing critical guidance for the industry.