Integrating metagenomics and metatranscriptomics into Orthoflavivirus diagnosis: a transformative approach for clinical virology

Diagnostic inaccuracies are a major yet often overlooked threat to global health, leading to delayed treatment, preventable harm and systemic gaps in disease control. Among the most affected domains are Orthoflavivirus infections, which pose ongoing diagnostic challenges due to antigenic cross-reactivity, overlapping clinical symptoms and the narrow temporal sensitivity of standard tools such as serology and reverse transcription polymerase chain reaction. These constraints have led to widespread misdiagnoses and underreporting, ultimately hampering both effective clinical management and public health response. Recent advances in metagenomic and metatranscriptomic sequencing offer a transformative solution by enabling unbiased, simultaneous pathogen detection and real-time profiling of viral and host transcriptomics. In this review, we assess the diagnostic performance and translational value of these approaches in resolving Orthoflavivirus infections, with case examples from clinical settings in countries like the USA, UK, China and Germany which have already implemented these approaches into routine diagnosis in some settings. We examine key methodological considerations, including optimal sample timing, sample types and processing, sequencing strategy selection and the diagnostic performance of various platforms. We highlight the growing use of metatranscriptomics for detecting active infections, profiling viral and host responses, identifying coinfections and supporting real-time surveillance. We also discuss the key challenges such as technical expertise, lack of standardization, cost, turnaround time and regulatory approval that currently limit global implementation. Finally, we highlight emerging international efforts to integrate sequencing-based diagnostics into routine hospital workflows. Together, these innovations mark a critical shift toward precision diagnostics for Orthoflavivirus infections, with broad implications for clinical settings.