ABSTRACT The type IV secretion system (T4SS) acts as the central virulence determinant of Brucella, facilitating intracellular survival via the secretion of effector proteins. In this study, we combined bioinformatic prediction with translocation assays to identify seven novel VirB-dependent effectors. Functional characterization revealed distinct roles for these proteins in both bacterial physiology and host-pathogen interactions. We identified BT4E19 and BT4E43 as critical determinants of cell envelope integrity: BT4E19 is required for core oligosaccharide maintenance and nitrosative stress tolerance, whereas BT4E43 is essential for O-antigen biosynthesis and oxidative stress resistance. Furthermore, BT4E43 is required for the efficient avoidance of lysosomal trafficking during intracellular infection. Notably, BT4E4 displays dual functions under tested conditions, being essential for oxidative stress resistance while simultaneously functioning to inhibit Caspase-5-mediated pyroptosis. In vivo assays further demonstrated that both BT4E19 and BT4E43 are indispensable for establishing chronic infection in mice. Collectively, these findings expand the Brucella effector repertoire and uncover the dual functions of specific effectors in maintaining bacterial structural integrity and orchestrating immune evasion, highlighting them as potential targets for anti-virulence therapies.
Schmallenberg virus (SBV) is a transboundary animal pathogen that causes reproductive disorders in ruminants, necessitating standardized molecular surveillance. Adhering to the Chinese national standard GB/T 43159—2023, we developed armored RNA quality control materials using MS2 bacteriophage technology targeting the conserved SBV S segment and comprehensively characterized their physicochemical properties. Transmission electron microscopy (TEM) showed icosahedral symmetric virus-like particles (VLPs, approximately 25 nm), with the stock concentration determined to be 3.48 × 1010 copies/mL via digital PCR (dPCR). The armored RNA control effectively withstood RNase A degradation and remained stable at 37 °C for over 30 days. In bovine serum matrix simulation assays at identical medium concentrations, the serum armored RNA group (Ct 26.57) was detected 4.89 cycles earlier than the degraded serum naked RNA group (Ct 31.46). This confirms the physical protective efficacy of the MS2 capsid. Concurrently, the serum armored RNA group showed a delay of only 2.17 cycles compared to the aqueous armored RNA group (Ct 24.40), exhibiting a typical matrix effect. Overall, this armored RNA enables full-process quality control encompassing extraction, reverse transcription, and amplification, providing a safe and stable technical reference to support molecular surveillance and diagnostic preparedness for cross-border SBV.
