Abstract:Ostreid herpesvirus 1 (OsHV-1) is a highly contagious pathogen responsible for mass mortality in various economically important bivalves, including oysters, posing a persistent threat to the global bivalve aquaculture industry. Its characteristic of latent infection makes early and accurate detection particularly crucial. Real-time quantitative PCR (qPCR), recommended by the World Organisation for Animal Health (WOAH), is the most widely used method for OsHV-1 detection. However, the accuracy of this technique and the comparability of results between different laboratories are highly dependent on the availability of traceable, homogeneous, and stable nucleic acid quality control materials (QCMs). Currently, such QCMs specifically for OsHV-1 are lacking. This gap directly leads to difficulties in direct comparison and mutual recognition of results from different detection systems, becoming a key constraint for advancing detection standardization, which is centered around laboratory proficiency testing and mutual recognition of results, as well as for implementing effective epidemic surveillance. Therefore, it is imperative to develop an OsHV-1 QCM that can simulate viral structure and is suitable for quality control throughout the entire process from nucleic acid extraction to amplification. Ferritin, with its engineerable nanocage structure—which provides exceptional stability, a controllable cavity for efficient encapsulation, and the ability to mimic viral architecture, thereby directly addressing the critical shortcomings of current platforms such as the unstable source material, demanding storage/transportation requirements, and biosafety risks associated with inactivated virus, the inherent inability to monitor the nucleic acid extraction process in synthetic nucleic acids like plasmids, and the inherent unsuitability for packaging DNA targets in virus-like particle systems such as MS2 bacteriophage—emerges as an ideal carrier for constructing such QCMs. This study aims to establish a preparation and evaluation system for an OsHV-1 nucleic acid detection QCM based on ferritin nanocages by engineering a positively charged interior cavity in ark clam (Scapharca broughtonii) ferritin and screening mutants for efficient loading of target DNA. Regarding the experimental methodology, this study first involved the rational design of critical negatively charged amino acid residues at the iron-nucleation sites within the cavity of S. broughtonii ferritin (SbFn). These residues were substituted with lysine or arginine to construct two interior mutants with a positively charged cavity, designated SbFn(+)Δ1 and SbFn(+)Δ2. The recombinant proteins were expressed, purified through heat precipitation to remove host proteins, followed by nickel-affinity chromatography and size-exclusion chromatography. The mutants were characterized using SDS-PAGE, native-PAGE, and transmission electron microscopy (TEM) to analyze subunit molecular weight, 24-mer assembly state, and nanocage morphology. A pH-mediated disassembly/reassembly strategy was employed to load the target OsHV-1 DNA fragment. Loading efficiency was assessed by measuring the molar ratio of DNA to protein, and DNase I digestion coupled with qPCR quantification was used to exclude external adsorption and confirm the encapsulation of DNA within the protein cavity. The optimal mutant was selected to prepare the QCM, designated NC-OsHV-1. Its characteristic value was assigned in strict accordance with the national standard (GB/T 15000.3-2023). Homogeneity was evaluated using one-way analysis of variance (F-test) to assess within-group and between-group variance, and stability was analyzed using a linear model for both short-term and long-term trends under conditions of 37°C, 4°C, –20°C, and –80°C. The results demonstrated that both constructed mutants, SbFn(+)Δ1 and SbFn(+)Δ2, successfully assembled into intact nanocages with a diameter of approximately 14 nm, retaining the reversible assembly capability of the wild-type protein. Loading experiments, assessed by the DNA-to-protein molar ratio, showed that the positively charged modification of the interior significantly enhanced DNA affinity, with SbFn(+)Δ2 exhibiting a 2.47 ± 0.10-fold increase in loading efficiency over the wild-type. DNase I digestion experiments confirmed that the vast majority of the target DNA was effectively encapsulated within the protein cavity. The characteristic value of the final NC-OsHV-1 QCM was determined to be (0.93 ± 0.12) × 10? copies/μL following rigorous qPCR analysis and statistical evaluation. Based on homogeneity testing (F=1.437) and stability trend analysis, the QCM was proven to possess excellent performance: no significant difference was found in within-group and between-group homogeneity; it remained stable for 7 days at 37°C, 21 days at 4°C, and at least 3 months at –20°C and –80°C, with no significant change in its characteristic value during these periods. In conclusion, this study successfully established an Ostreid herpesvirus 1 (OsHV-1) nucleic acid detection QCM and its preparation system based on ferritin nanocages. The developed QCM exhibits good homogeneity, stability across a wide temperature range, and accurate assigned value. It can simulate viral samples and is suitable for full-process quality control from nucleic acid extraction to amplification. This achievement provides not only a key technical tool and solution for standardizing OsHV-1 nucleic acid detection but also offers a novel and referable methodology for developing nucleic acid reference materials for other aquatic pathogens. Ultimately, it will provide effective support for the standardization and capability enhancement of China's aquatic animal disease surveillance network.
