Porcine reproductive and respiratory syndrome virus (PRRSV) is globally prevalent and seriously harms the economic efficiency of pig farming. Because of its immunosuppression and high incidence of mutant recombination, PRRSV poses a great challenge for disease prevention and control. Nonstructural protein 2 (NSP2) is the most variable functional protein in the PRRSV genome and can generate NSP2N and NSP2TF variants due to programmed ribosomal frameshifts. These variants are broad and complex in function and play key roles in numerous aspects of viral protein maturation, viral particle assembly, regulation of immunity, autophagy, apoptosis, cell cycle and cell morphology. In this paper, we review the structural composition, programmed ribosomal frameshift and biological properties of NSP2 to facilitate basic research on PRRSV and to provide theoretical support for disease prevention and control and therapeutic drug development.

Currently, multiple recombinant variants of porcine reproductive and respiratory syndrome virus (PRRSV) are circulating in China. These variants exhibit differences in pathogenicity. To comprehensively understand the current status of the virus and its recombination patterns, a total of 677 PRRSV strains were used for evolutionary analysis, of which 673 strains were isolated from mainland China. The results indicated that current PRRSV strains in circulation in China are highly complex, with NADC30‐like strains having replaced highly pathogenic strain of PRRSV (HP‐PRRSV) strains as the dominant strains. An analysis of the patterns and hotspots of PRRSV‐2 recombinant strains from 2019 to 2023 revealed two main types: the strains with NADC30 as the primary parent were observed to have recombination hotspots concentrated in the Nsp1, Nsp4 to Nsp9, and ORF2 to ORF6 regions, while recombination frequencies were found to be lower in the Nsp2, Nsp10, and Nsp11 regions. In contrast, the other type of recombinant strain, primarily derived from HP‐PRRSV, exhibited recombination hotspots concentrated in the Nsp2 and ORF2 to ORF6 regions, while the recombination frequency in the Nsp8 and Nsp9 regions was relatively low. Further, a comprehensive analysis of the phylogenetic and recombination patterns of four PRRSV strains (ZJ‐2021‐1, JS‐2020‐1, GZ‐2022‐1, and SH‐2020‐2) indicated variations in genotyping precision among Nsp2, ORF5, and the entire genome. As demonstrated by the results of the animal experiments, there were differences in pathogenicity between ZJ‐2021‐1 and JS‐2020‐1. The pathogenicity of the recombinant strain exhibited similar characteristics to its parental skeleton. Our results provided reference data for exploring epidemiological characteristics and pathogenicity of recombinant PRRSV strains.

With the global prevalence of pigeon racing, pigeon infections with Pigeon circovirus (PiCV) have been reported across the world. PiCV-infected pigeons are susceptible to subsequent infections and exhibit a range of symptoms including weight loss, lethargy, anorexia, respiratory distress and diarrhea, which exerts a huge impact on the pigeon industry. However, there is currently a lack of epidemiological data on PiCV infection in pigeons. Therefore, the aim of this study was to gain a comprehensive understanding of the prevalence, genetic variations, and evolutionary dynamics of PiCV within pigeon populations. In this study, we collected 28 samples from four cities in China to assess the prevalence of PiCV. The results showed that the positive rate was 92.86%, which indicated that PiCV was widespread in Chinese pigeon flocks. Meanwhile, whole genome sequences of PiCV were obtained for partial positive samples. The results showed that there were 12 different clades of PiCV, and the samples collected in this study have been classified as types 1, 4, 6, and 11. The results of the PiCV genome analysis indicate a high incidence of recombination events. Further analysis of the encoded Cap protein at the gene and protein level revealed a high degree of variation in the Cap protein. The amino acids at positions 30-120 of the Cap protein exhibited the greatest frequency of variation. Meanwhile, we found that amino acid sequences 140-180 were relatively conserved, which was related to the immunogenicity of the circovirus Cap protein. The results of the antigenicity analysis demonstrated that amino acid sequences 140-180 exhibited strong antigenicity, indicating the potential of cap to serve as a candidate protein in the production of a PiCV vaccine. The present study provided epidemiological information on PiCV, which promises to facilitate the development of effective control measures to prevent its transmission.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen posed a serious threat to the global swine industry. In this study, a BAC-based reverse genetics platform was established using a highly pathogenic PRRSV (HP-PRRSV) strain. Three recombinant reporter viruses were constructed by inserting the enhanced green fluorescent protein (EGFP) gene into three different intergenic regions of the complete PRRSV-L251 genome. Immunofluorescence assays combined with viral growth kinetics and reporter gene stability assessments indicated that rL251-ORF4-5a-EGFP maintained relatively stable expression during serial passage, and viral titers at 72 hours post-infection (hpi) were comparable to the parental virus. Subsequently, we identified four candidate compounds with potential anti-PRRSV activity using rPRRSV-L251-ORF4-5a-EGFP, indicating that this platform can be used as a visual assessment tool for antiviral drug screening. This study demonstrated that the ORF4-5a interval region is a feasible and promising site for exogenous gene insertion, and provided a robust technical platform for PRRSV vaccine development and pathogenesis studies.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to the swine industry worldwide. The viral matrix (M) protein is essential for virion assembly and infectivity. In this study, we found that the M protein is degraded through the ubiquitin-proteasome system. Immunoprecipitation and mass spectrometry identified the host deubiquitinase OTUB1 as an M-binding partner. Overexpression of OTUB1 increased M protein stability and expression levels, while OTUB1 knockdown promoted M degradation. OTUB1 interacted with the C-terminal ectodomain of the M protein and specifically removed K48-linked ubiquitin chains that target proteins for proteasomal degradation. Mechanistically, a catalytically inactive OTUB1 mutant (C91A) retained the ability to stabilize and deubiquitinate M, while a mutant defective in the non-canonical pathway (D88A) lost this function, indicating that OTUB1 acts primarily by sequestering E2 ubiquitin-conjugating enzymes. Furthermore, we identified UBE2D2 as the specific E2 ubiquitin-conjugating enzyme sequestered by OTUB1 to prevent M protein degradation. Depletion of OTUB1 significantly attenuated PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages. OTUB1 specifically targeted the M protein without stabilizing other PRRSV proteins. Importantly, OTUB1-mediated stabilization was conserved across M proteins from diverse PRRSV strains, including PRRSV-1, PRRSV-2, and emerging NADC30-like and NADC34-like variants. Collectively, our findings revealed a host mechanism exploited by PRRSV to enhance replication and identified OTUB1 as a potential target for antiviral development. IMPORTANCE: Porcine reproductive and respiratory syndrome (PRRS) poses a substantial threat to global swine production, resulting in major economic losses. The causative agent, PRRS virus (PRRSV), exhibits high genetic variability, which frequently enables it to evade existing vaccine-induced immunity. Our study identifies the host deubiquitinase OTUB1 as a critical factor exploited by PRRSV to stabilize the essential M protein by sequestering the E2 ubiquitin-conjugating enzyme UBE2D2. This mechanism facilitates enhanced viral replication and is conserved across diverse PRRSV strains. Our findings reveal a key host-pathogen interaction and suggest that disrupting OTUB1-mediated stabilization of the M protein may pave the way for novel antiviral strategies against diverse PRRSV variants. This work thus provides a conceptual foundation for future host-directed interventions against this economically significant pathogen.

Porcine circovirus 3 (PCV3) is an emerging swine pathogen associated with reproductive disorders, multisystemic inflammation, and other clinical syndromes. The capsid protein (Cap), the principal structural and immunogenic component, is an ideal antigen for serological detection. However, its poor solubility in prokaryotic expression systems limits diagnostic assay development. In this study, the N-terminal 32 amino acids of Cap were truncated, and the protein was expressed in a soluble form using the SHuffle T7 Express strain. An indirect enzyme-linked immunosorbent assay (ELISA) using purified soluble Cap as a coating antigen was established and optimized. The assay exhibited excellent specificity, with no cross-reactivity against other common swine pathogens, high sensitivity, and good repeatability, with intra- and inter-assay coefficients of variation below 10%. The cut-off value was determined, and testing of 144 porcine serum samples showed a 78.47% concordance rate with PCR, indicating good diagnostic performance. This Cap-based indirect ELISA provides a reliable, cost-effective, and high-throughput tool for large-scale serological surveillance and epidemiological monitoring of PCV3, supporting improved disease control strategies in pig populations.