Identification and Detection of Novel Animal Viruses: A Genome-First Surveillance and CRISPR Diagnostic Workflow

Emerging animal viruses threaten livestock and human health, yet many circulate undetected because most diagnostics target known agents. We evaluated an end-to-end workflow for rapid identification and field-ready detection of novel animal viruses by conducting cross-sectional surveillance at 18 livestock-wildlife-peri-domestic sites. Oropharyngeal, rectal, and serum samples (n=1,964) underwent metatranscriptomic sequencing (Illumina/Nanopore) and pan-viral consensus PCR, with read quality control, host depletion, and taxonomic assignment using k-mer based classifiers, de novo assembly, and protein-level homology searches. Novelty was defined as <90% amino acid identity in conserved proteins; relationships and divergence were inferred with maximum-likelihood and relaxed-clock phylogenies. For priority clades, we designed CRISPR-Cas12 and SYBR qPCR assays and assessed analytical sensitivity, cross-reactivity, and blinded diagnostic performance. We detected sequences from 312 viral taxa across 24 families; 27 lineages met novelty criteria. Ten near-complete genomes were recovered (median coverage 47x). Newly identified paramyxoviruses clustered within Orthorubulavirus (fruit bats), and two coronaviruses formed distinct subclades within Alphacoronavirus. CRISPR assays for four representatives achieved limits of detection of 10–100 copies per uL with no cross-reactivity to 42 heterologous viruses; in blinded panels, sensitivity was 93–97% and specificity 98–100% (AUC 0.96). These findings show that a genome-first surveillance pipeline coupled to rapidly deployable CRISPR diagnostics can reveal and confirm previously unrecognized animal viruses with high accuracy; integration into routine One Health monitoring could accelerate risk assessment, guide targeted mitigation, and narrow the window between viral emergence and detection.