High Recombination and Intra-oocyst Heterogeneity Shape Virulence in Cryptosporidium parvum, with CpDHR1 Defined as a Secondary Virulence Determinant

Cryptosporidium parvum causes life-threatening diarrhea in infants and neonatal animals, with genetic recombination driving virulence variation, yet intra-oocyst genomic heterogeneity remains uncharacterized. Here, we crossed fluorescently tagged virulent IIdA20G1-HLJ and avirulent IIdA19G1-GD strains in IFN-γ knockout mice, performing whole-genome sequencing of single oocysts/sporozoites and establishing single-oocyst expansion lines. We found extreme intra-oocyst genomic heterozygosity, with sporozoites harboring mosaic parental alleles due to frequent crossovers. A high-resolution genetic map revealed an 8.3 kb/cM recombination rate with telomere-biased crossovers and 28 recombination hotspots near infectivity-associated genes. Expansion lines showed divergent virulence unlinked to gp60, and integrative comparative genomics identified cgd5_4090 (CpDHR1), a DEAH-box RNA helicase, as a secondary virulence factor. Revsere genetic studies confirmed that CpDHR1 modulates parasite growth and virulence, possibly via effects on ribosome assembly due to conformational flexibility. These findings shed light on the evolution of C. parvum virulence and offer novel targets for cryptosporidiosis. control.