Tilapia lake virus (TiLV) poses a major challenge to tilapia aquaculture. Identifying host factors that support viral replication can improve understanding of disease susceptibility and inform genetic approaches to disease resistance. This study used CRISPR/Cas9 genome editing to investigate the contribution of acidic nuclear phosphoprotein 32 family member A (ANP32A) to TiLV infection in a Mozambique tilapia brain (OmB) cell line.
An optimised ribonucleoprotein-based CRISPR/Cas9 editing protocol was used to disrupt anp32a, followed by single-cell cloning. Sanger sequencing identified a single-nucleotide insertion in exon 3, introducing a frameshift and a premature stop codon. The edited clone and wild-type cells were challenged with TiLV at a multiplicity of infection of 0.01. Viral replication was evaluated by RT-qPCR and infectious virus titration, alongside assessment of cytopathic effects. Transcriptomic and proteomic analyses were integrated to characterise cellular changes associated with anp32a disruption under uninfected and infected conditions.
Compared with wild type cells, the anp32a disrupted clone exhibited reduced viral RNA abundance and less pronounced cytopathic effects. Infectious virus titres were approximately tenfold lower at 12 days post-infection, indicating reduced, but not abolished, viral replication. Integrated transcriptomic and proteomic analyses revealed changes in pathways associated with mitochondrial function, energy metabolism, aminoacyl-tRNA biosynthesis and protein translation. Concordant reductions in genes and proteins involved in these processes suggest that impaired metabolic and biosynthetic capacity may contribute to the reduced ability of edited cells to support TiLV replication.
These findings support a proviral role for ANP32A in tilapia cells and demonstrate the utility of CRISPR/Cas9 editing for investigating host determinants of TiLV susceptibility. ANP32A represents a candidate for further investigation into genetic approaches to TiLV resistance. Validation in additional independently edited clones and in vivo studies will be needed to establish the reproducibility of the phenotype and assess potential consequences for fish growth and health.