Master of Science, The Ohio State University, 2022, Veterinary Preventive Medicine

Rotavirus A (RVA) is the primary cause of acute viral gastroenteritis in children and young animals globally; however, its replication and pathogenesis remain poorly understood. We have previously demonstrated contrasting modes of interactions with the host cell glycans for two prevalent porcine RVA strains: OSU G5P[7] (historically associated with severe disease in piglets) and G9P[13] (globally emerging variant in humans and swine). Specifically, OSU G5P[7] and G9P[13] strain replication was significantly decreased and significantly increased, respectively, following removal of terminal sialic acids (SA) by neuraminidase (NA) treatment. The latter contrasting effects coincided with the presence of distinct mutations found in the VP4 fusion region of these strains. In our first study, to clarify cellular mechanisms associated with these differential mechanisms of cell attachment/entry we conducted transcriptome analysis of porcine small intestinal enteroids (PIEs) infected with the two RVA strains with and without NA treatment. NA treatment of porcine intestinal enteroids alone, before individual RVA G9P[13]/OSU G5P[7] infection resulted in altered expression of genes associated with biological regulation, transporter activity, protein binding, and multicellular organismal processes. This was shown with significant contradicting impacts, with G9P[13] being significantly enhanced, and OSU G5P[7] replication being significantly inhibited. Cholesterol (a key component of the host plasma membranes) has been shown to play a critical role in RVA replication. To further improve our understanding of RVA pathogenesis taking into consideration RVA genotype-specific features, in our second study, we comparatively evaluated the effects of cholesterol and cholesterol-related additives [Methyl-β-cyclodextrin (MβCD), and diethylaminoethyl (DEAE), and bile acids (BAs)] on G9P[13] vs. OSU G5P[7] replication in vitro. Consistent with our previous findings, treatment with cholest (open full item for complete abstract)

Committee: Anastasia Vlasova (Advisor); Linda Saif (Committee Member); Qiuhong Wang (Committee Member) Subjects: Biology; Genetics; Virology

Doctor of Philosophy, The Ohio State University, 2021, Comparative and Veterinary Medicine

Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Cell glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established porcine small intestinal enteroids (PIEs) expressing different HBGAs (A+, H+, and A+/H+) to study RV-glycan interactions. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] and PRV OSU G5P[7] virulent and attenuated strains. Virulent Wa replicated to the highest titers in A+ PIEs, whereas virulent PRV G9P[13] and OSU strains preferentially replicated in H+ PIEs. The replication of all 4 attenuated strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for Wa replication; however, they seemed dispensable for G9P[13] and OSU strains. Contrasting outcomes were observed following sialidase treatment on PIEs which significantly enhanced G9P[13] replication, but inhibited OSU G5P[7] growth. These observations suggest that additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. To study the molecular mechanisms of RV attenuation and cell culture adaptation, we have sequenced and analyzed complete genomes of virulent Wa, (G1P[8]), M (G3P[8]), Gottfried (G4P[6]) and OSU (G5P[7]) strains and their attenuated counterparts. Most of the mutations clustered in the VP4 gene of the attenuated strains, with a high nonsynonymous substitution rate (81.2%). Two amino acid (aa) substitutions, aa385 and aa 471, found in the VP4 gene were conserved between two or more strain pairs. Of interest, the D393H and D385N substitutions identified within the VP4 hydrophobic domain important for virus cell entry. Collectively, these (open full item for complete abstract)

Committee: Vlasova Anastasia Dr. (Advisor); Scott P Kenney Dr. (Committee Member); Qiuhong Wang Dr. (Committee Member); Linda J Saif Dr. (Committee Member) Subjects: Biology; Virology