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

Authors: Alvarez Narvaez, Sonsiray; Harrell, Telvin; SELLERS, HOLLY - Ceva Animal Health; MARKIS, MILOS - Aviserve, Llc; Conrad, Steven

Submitted to: Journal of Applied Poultry Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2026
Publication Date: 7/2/2026
Citation: Alvarez Narvaez, S., Harrell, T.L., Sellers, H., Markis, M., Conrad, S.J. 2026. Direct comparison of footpad inoculation with oculo-nasal inoculation for the assessment of avian reovirus virulence. Journal of Applied Poultry Research. https://doi.org/10.1016/j.japr.2026.100761.
DOI: https://doi.org/10.1016/j.japr.2026.100761

Interpretive Summary: The avian reoviruses infect chickens, turkeys, and other poultry animals. Compared to other poultry diseases, the amount of research done on the avian reoviruses to date is fairly small. Because avian reoviruses are a somewhat new area of research, many of the tools used for researching other poultry viruses do not yet exist. One such tool is assays to tell how severe (virulent) a particular strain of avian reovirus is. In this paper we describe a test for virulence that can be used to rate and rank avian reoviruses.

Technical Abstract: Avian orthoreoviruses (ARVs) are economically significant pathogens of poultry associated with viral arthritis, enteric disease, malabsorption syndrome, runting and stunting syndrome (RSS) and reduced flock uniformity. 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 tissue tropism of genetically diverse ARV isolates using two inoculation routes, the oculo-nasal route and the footpad route. Six ARV strains representing 5 sC-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 5x10^4 plaque-forming units (pfu) via either the footpad or ocular–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 pathogenicity among strains and allowing classification into four virulence categories ranging from highly virulent to avirulent. Furthermore, strain-dependent differences in viral tropism were observed after footpad inoculation, with the spleen emerging as the preferred site of replication for all virulent isolates. In contrast, ocular–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 pathogenicity assessment and provide new insights into strain-dependent virulence and tissue tropism.