1 Abstract 2 Rotaviruses (RVs) of group A (RVAs) remain an important cause of acute gastroenteritis and mortality in young animals 3 and children. Despite their significance, mechanisms of RVA cell entry, replication and attenuation remain poorly 4 understood indicating a critical need to address these knowledge gaps and design universal control approaches. 5 We have previously conducted comparative sequence analysis that identified two key amino acid substitutions 6 associated with cell culture adaptation and attenuation: D385N in the VP4 of most human and porcine RVA strains and 7 D393H in porcine OSU strain VP4 only. While these or similar mutations were previously suggested to be important for 8 cell culture adaptation/attenuation, their function has not been confirmed experimentally. Using our porcine intestinal 9 enteroid (PIE) system, we have demonstrated that contrasting modes of interactions with the host glycans determine 10 replication efficacy of different RVA strains. OSU replication was uniquely and significantly down-regulated by the 11 removal of terminal sialic acids, while the latter significantly enhanced the replication of a novel porcine G9P[13] strain. 12 OSU is a historic dominant RVA variant characterized by robust in vivo and in vitro replication and consistently 13 associated with diarrheal disease in piglets for several decades, while G9P[13] and G9P[19] are recent, globally 14 emerging RVA variants in swine and humans. It is possible that the latter strains have evolved to acquire some biological 15 advantage allowing for their efficient spread and replication in different hosts. Virulent OSU and G9P[13] strains have 16 unique aa substitutions in the aa positions 385 and 393 of the VP4 hydrophobic loop, which could be a reason for 17 variable interactions with host glycans. Additionally, our recent study demonstrated that the host (PIE) transcriptome 18 response associated with OSU and G9P[13] infections differed drastically altering glycan expression, cholesterol 19 metabolism and innate immune signaling in a strain-specific manner. These findings suggest that evolutionary 20 adaptation of RVAs to the host and host glycans is a critical mechanism allowing for their interspecies (including 21 zoonotic) transmission. 22 Following a previously optimized protocol, our lab has successfully cloned 11 segments of virulent RVA OSU into the 23 pT7 plasmid and rescued infectious virus. We now propose to use this reverse genetics system for in-depth exploration 24 of the molecular mechanisms of RVA virulence, cell attachment and replication. The specific aims to achieve this goal 25 are summarized below: 26 Aim 1: Evaluate if the D385N and D393H substitutions (individually or in combination with other mutations in the OSU 27 VP4) introduced into OSU RGS (ic-virOSU) will confer an attenuated phenotype to the progeny virus (ic-attOSU). Aim 28 2: Evaluate if G9P[13]-like mutations (S385N and D393N) will confer the G9P[13]-l...