Genetic crosses of Cryptosporidium parvum isolates produce progeny with heterogeneous genomes and varying virulence

Cryptosporidium is a leading cause of diarrhea in infants and neonatal animals worldwide, resulting in stunted growth and increased mortality. Evidence suggests that genetic recombination between subtypes and adaptive selection can lead to the emergence of variants with altered virulence. In this study, we used whole-genome sequencing and comparative genomic analysis to characterize the genetic heterogeneity within single oocysts resulting from genetic crosses between two strains of different virulence. Each oocyst contained sporozoites with distinct genomes, and expanded lines of single oocysts exhibited significant differences in infection patterns and virulence. Through comparative genomic analyses of the expanded lines and experimental validation, we identified a DEAH-box RNA helicase (CpDHR1), a homolog of the well-known small subunit ribosome assembly regulator, as a secondary virulence factor. These results help us to understand the evolution and regulation of virulence in Cryptosporidium and guide the development of surveillance systems and control measures.