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Summary: The health of the Western honey bee is threatened by a global epidemic of deformed wing virus (DWV) infections driven by the ectoparasitic mite Varroa destructor acting as mechanical and biological virus vector. Three different variants of DWV, DWV‐A, ‐B and ‐C exist. Virulence differences between these variants and their relation to V. destructor are still controversially discussed. We performed laboratory experiments to analyze the virulence of DWV directly isolated from crippled bees (DWVP0) or after one additional passage in bee pupae (DWVP1). We demonstrated that DWVP0 was more virulent than DWVP1 for pupae, when pupal mortality was taken as virulence marker, and for adult bees, when neurotropism and cognitive impairment were taken as virulence markers. Phylogenetic analysis supported that DWV exists as quasispecies and showed that DWVP0 clustered with DWV‐B and DWVP1 with DWV‐A when the phylogeny was based on the master sequences of the RNA‐dependent RNA polymerase but not so when it was based on the VP3 region master sequences. We propose that switching of DWV between the bee and the mite host is accompanied by changes in viral sequence, tissue tropism and virulence and that the RNA‐dependent RNA polymerase is involved in determining host range and virulence. [ABSTRACT FROM AUTHOR]