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Senecavirus A (SVA) is a newly emerging picornavirus associated with swine vesicular lesions and neonatal mortality, threatening the global pig industry. Despite sustained efforts, the molecular mechanisms of SVA pathogenesis have not yet been fully elucidated. Here, we demonstrate for the first time that SVA infection can induce complete mitophagy in host cells, which depends on SVA replication. Mitophagy has been subsequently proven to promote SVA replication in host cells. Genome-wide screening of SVA proteins involved in inducing mitophagy showed that although VP2, VP3, 2C, and 3A proteins can independently induce mitophagy, only the 2C protein mediates mitophagy through direct interaction with TUFM (Tu translation elongation factor, mitochondrial). The glutamic acids at positions 196 and 211 of TUFM were shown to be two key sites for its interaction with 2C protein. Moreover, TUFM was discovered to interact directly with BECN1 and indirectly with the ATG12–ATG5 conjugate. Further experiments revealed that TUFM needs to undergo ubiquitination modification before being recognized by the macroautophagy/autophagy receptor protein SQSTM1/p62, and E3 ubiquitin ligase RNF185 catalyzes K27-linked polyubiquitination of TUFM through the interaction between RNF185's transmembrane domain 1 and TUFM to initiate SVA-induced mitophagy. The ubiquitinated TUFM is recognized and bound by SQSTM1, which in turn interacts with MAP1LC3/LC3, thereby linking the 2C-anchored mitochondria to the phagophore for sequestration into mitophagosomes, which ultimately fuse with lysosomes to achieve complete mitophagy. Overall, our results elucidated the molecular mechanism by which SVA induces mitophagy to promote self-replication and provide new insights into SVA pathogenesis.Abbreviations: aa: amino acid; Baf A1: bafilomycin A1; BHK-21: baby hamster kidney-21; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GST: glutathione S-transferase; HA: hemagglutinin; hpi: hours post-infection; hpt: hours post-transfection; IPTG: isopropyl β-D-1-thiogalactopyranoside; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor-1; MOI: multiplicity of infection; mRFP: monomeric red fluorescent protein; MS: mass spectrometry; ORF: open reading frame; PBS: phosphate-buffered saline; SD: standard deviation; SQSTM1/p62: sequestosome 1; ST: swine testis; SVA: Senecavirus A; TCID50: 50% tissue culture infectious dose; TIMM23: translocase of inner mitochondrial membrane 23; TM: transmembrane; TOMM20: translocase of outer mitochondrial membrane 20; TUFM: Tu translation elongation factor, mitochondrial; Ub: ubiquitin; UV: ultraviolet; VDAC1: voltage dependent anion channel 1; WT: wild-type; μg: microgram; μm: micrometer; μM: micromole. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome virus (PRRSV) has significantly impacted the global pork industry for over three decades. Its high mutation rates and frequent recombination greatly intensifies its epidemic and threat. To explore the fidelity characterization of Chinese highly pathogenic PRRSV JXwn06 and the NADC30-like strain CHsx1401, self-recombination and mutation in PAMs, MARC-145 cells, and pigs were assessed. In vitro, CHsx1401 displayed a higher frequency of recombination junctions and a greater diversity of junction types than JXwn06. In vivo, CHsx1401 exhibited fewer junction types yet maintained a higher junction frequency. Notably, JXwn06 showed more accumulation of mutations. To pinpoint the genomic regions influencing their fidelity, chimeric viruses were constructed, with the exchanged nsp9-10 regions between JXwn06 and CHsx1401. The SJn9n10 strain, which incorporates JXwn06's nsp9-10 into the CHsx1401 genome, demonstrated reduced sensitivity to nucleotide analogs compared to CHsx1401. Conversely, compared with JXwn06, the JSn9n10 strain showed increased sensitivity to these inhibitors. The swapped nsp9-10 also influences the junction frequency and accumulated mutations as their donor strains. The results indicate a propensity for different types of genetic variations between these two strains and further highlight the nsp9-10 region as a critical determinant of their fidelity. [ABSTRACT FROM AUTHOR]

Glaesserella parasuis is a specific bacterial pathogen of Glässer's disease which causes significant economic losses to the swine industry. Dependable and rapid detection of G. parasuis is crucial to prevent and control Glässer's disease outbreaks. In this study, a recombinase-aided amplification (RAA) assay based on the infB gene was developed to rapid detect G. parasuis. The novel method performs isothermal detection at 42°C for real-time analysis or visualization and data analysis (RAA-VDA). The developed assay showed high specificity for G. parasuis detection without cross-reactions to other clinically important swine pathogens. The analytical sensitivity of real-time RAA was 67.17 copies per reaction with 95% reliability, which was comparable to the G. parasuis quantitative real-time PCR (qPCR). However, the detection limit of RAA-VDA was 142.43 copies per reaction with 95% reliability. The coefficient of variation analysis of the intrabatch and interbatch experimental replicate results were less than 4.30% and 6.74%, respectively, indicating the real-time RAA assay had high repeatability and reproducibility. A total of 108 clinical tissue samples were used to evaluate the clinical diagnostic performance. The diagnostic accordance rates of qPCR with real-time RAA and RAA-VDA were 100% and 98.15% (106/108), respectively. This system combined instrumental analysis and visualized analysis to accomplish a new try for rapid detection of G. parasuis in clinical practice. [ABSTRACT FROM AUTHOR]

Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV), which has caused great economic losses to the pig industry worldwide. Reliable and timely diagnose is crucial for the surveillance, control and eradication of PR. Here, a real‐time fluorescent recombinase‐aided amplification (real‐time RAA) assay was established to detect PRV. Primers and probes were designed based on the conserved regions of the PRV gE gene. The assay was specific for the detection of wild‐type PRV, showing no cross‐reactivity with other important porcine viruses (including PRV gE‐deleted vaccine strains). Analytical sensitivity of the assay was three 50% tissue culture infectious doses (TCID50) of PRV DNA per reaction with 95% reliability, which is comparable to that of a PRV‐specific real‐time PCR (qPCR) assay. In diagnosis of 206 clinical tissue samples, the diagnose accordance rate between the real‐time RAA assay and qPCR assay was 97.57% (201/206). Interestingly, the amplified products of real‐time RAA could be visualized under a portable blue light instrument, making it possible for the rapid detection of PRV in resource‐limited settings and on‐site screening. Therefore, our developed real‐time RAA assay is a diagnostic method for the rapid detection of PRV in the field. [ABSTRACT FROM AUTHOR]