ABSTRACTWe conducted research studies on avian reovirus (ARV) infectivity in egg-laying hens, focusing on three variants (δC genotypes 2, 3, and 5) detected in layer chickens in Pennsylvania to date. Day-old chicks (Hy-Line North America, LLC, PA), raised at the Poultry Education and Research Center of Penn State University Park campus, showed healthy growth and normal egg production after 20 weeks of age. ARV variants were propagated in Leghorn male-chicken hepatocellular-carcinoma cell cultures, with concentrations measured at TCID50/ml. Each group of 10 hens received a 1.0 ml dose containing 103-104 TCID50/ml of one ARV variant through oral, nasal, and ocular routes. Infected hens showed normal egg production, with minimal signs of watery droppings in the first-week post-inoculation (pi). Cloacal and oral pharyngeal swabs were collected daily in the first week pi and every other day in the second-week pi to monitor virus shedding. Virus shedding began 24 h pi through faeces, peaked at 2-4 days pi, decreased by 5-7 days pi, and ceased after 12-14 days pi. A few birds' oral pharyngeal swabs were weakly positive for 1-3 days pi, then all turned negative. Infected hens developed high serum and egg yolk antibody titres at 2-3 weeks pi, showing 100% protection against subsequent infections with the same variant strain, demonstrating a 100% protection rate.RESEARCH HIGHLIGHTSAvian reovirus-infected hens shed virus heavily at 2-3 days post-inoculation.Shedding became minimal after 5-7 days post-inoculation.ARV variants offered 100% protection in hens upon subsequent infections.Infected hens maintained normal egg production with no observable clinical signs.