Discrimination of Epstein–Barr virus latent infection and lytic replication reactivation using a novel methyl-CRISPR biosensor Free

Introduction:

Epstein–Barr virus (EBV), the first identified human oncogenic gamma-herpesvirus, establishes lifelong persistent infection through a dual-phase viral life cycle: latent infection and lytic replication. Studies have shown that DNA methylation inhibits the activity of gene promoters through CpG site methylation, which is the core epigenetic mechanism by which EBV regulates the latent/lytic state. The viral genome is highly methylated at specific sites in EBV-infected cancer cells and latent infection cell lines, whereas the methylation level is lower in cells undergoing lytic replication and viral particles. Therefore, methylation profiling of EBV genomes can distinguish viral states: low methylation correlates with lytic replication, and high methylation indicates latency. Despite this mechanistic insight, no direct, cost-effective, or technically simple method exists to distinguish the latent infection EBV genome and the lytic replication state.

Methods: We have established an ultrasensitive platform for the detection of DNA methylation utilizing RPA-mediated CRISPR/Cas13a technology. This innovative strategy synergistically integrates the high specificity of BstUI/HhaI restriction endonucleases in the digestion of unmethylated cytosines with the robust signal amplification efficiencies of the RPA and CRISPR/Cas13a systems. This activated Cas13a indiscriminately degrades nearby fluorescent RNA reporters, thereby enabling sensitive detection of DNA methylation events. The EBV-positive cell line Raji (in a latent cycle) was treated with drugs and then reactivated to enter the replication cycle. DNA within the cells before and after treatment was collected and quantitatively analyzed directly using a methyl-sensitive CRISPR biosensor. The clinical samples used were as follows: EBV-positive saliva from healthy subjects (n=10) and whole blood from patients with primary EBV infection (n=9).

Results: To validate the feasibility of the methyl-CRISPR biosensor, we initially employed model double-stranded DNA constructs with and without methylation to demonstrate that digestion with BstUI/HhaI enzymes was feasible and could rapidly trigger RPA. We subsequently optimized the reaction components and time parameters, confirming its ability to detect methylation-specific targets within 0.5 h. Furthermore, the practical applicability of the method was evaluated using serum samples from EBV-infected individuals, which yielded acceptable results, with recovery rates ranging from 91.3% to 96.9%. Collectively, these findings unequivocally demonstrate that the methyl-CRISPR biosensor provides a viable solution for high-sensitivity DNA methylation analysis while also proving effective for methylation detection in practical clinical applications.

Following validation through DNA modeling, we selected four EBV genomic loci (BZLF1, BALF5, LF2, and BDLF2) harboring CCGG motifs for comparative analysis of their methylation status. The fluorescence signal intensity ratios for the methylation markers in the nonactivated cells were 1000-fold higher than those in the reactivated samples across all four loci. This differential methylation pattern, detectable via methyl-CRISPR biosensor-based targeting, demonstrates the ability of this system to quantify latent versus lytic EBV DNA proportions in infected cells. These results underscore the utility of locus-specific methylation profiling as a biomarker for viral replication state discrimination in oncogenic herpesvirus research.

Blood samples from nine patients with primary EBV infection were analyzed. Most of the tested genomic regions of EBV exhibited high methylation levels, indicating that the majority of the EBV genomes detected in the peripheral blood during primary infection were in a latent state. In PTLD patients, elevated methylation indices across multiple EBV genomic regions further confirmed the presence of latent EBV DNA. This finding suggests that the observed high viral loads in these patients result from the proliferation of cells harboring latent viral genomes.

Conclusions: Overall, our study demonstrates that the methyl-CRISPR biosensor offers a convenient methodology for discriminating between latent and reactivation EBV genomes within biological specimens. We propose that this approach enables rapid, facile, and cost-effective differentiation of the EBV latency status, thereby providing a valuable basis for guiding clinical management and therapeutic strategies.