Development, Characterization, and Validation of an MS2 Phage-Based Armored RNA Control for Schmallenberg Virus...

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.