Direct comparison of footpad inoculation with oculo-nasal inoculation for the assessment of avian reovirus virulence

Orthoreovirus avis or avian reoviruses (ARVs) are economically significant pathogens of poultry. Despite their importance, standardized experimental models for assessing ARV virulence remain limited, complicating comparisons among field isolates and across laboratories. In this study, we developed and evaluated an infection model to rank the virulence and investigate the organ distribution patterns of genetically diverse ARV isolates using two inoculation routes, the oculo-nasal route and the footpad route. Six ARV strains representing 5 σC-based genoclusters were verified as single-strain preparations by whole-genome sequencing and quantified by plaque assay prior to inoculation. One-day-old specific pathogen-free (SPF) White Rock chicks were inoculated with 5 × 10 4 plaque-forming units (pfu) via either the footpad or oculo–nasal route and monitored for clinical signs, body weight, and survival. Viral RNA loads were quantified in multiple organs using RT-qPCR. Footpad inoculation consistently induced clinical disease and mortality, enabling clear differentiation of virulence among strains and allowing classification into three virulence categories: highly virulent, virulent and avirulent. Furthermore, strain-dependent differences in organ distribution were observed after footpad inoculation, with the spleen emerging as the site with the highest viral RNA detection for all virulent isolates. In contrast, oculo–nasal inoculation with an amount of virus equal to that used in footpad inoculation resulted in minimal clinical disease and mortality, despite evidence of infection, limiting its utility for virulence ranking. These findings establish footpad inoculation combined with an exact viral quantification via plaque assay as a robust and scalable model for comparative ARV virulence assessment and provide new insights into strain-dependent virulence and tissue tropism.