Differential transcriptome response following infection of porcine ileal enteroids with species A and C rotaviruses.

BACKGROUND: Rotavirus C (RVC) is the major causative agent of acute gastroenteritis in suckling piglets, while most RVAs mostly affect weaned animals. Besides, while most RVA strains can be propagated in MA-104 and other continuous cell lines, attempts to isolate and culture RVC strains remain largely unsuccessful. The host factors associated with these unique RVC characteristics remain unknown. METHODS: In this study, we have comparatively evaluated transcriptome responses of porcine ileal enteroids infected with RVC G1P[1] and two RVA strains (G9P[13] and G5P[7]) with a focus on innate immunity and virus-host receptor interactions. RESULTS: The analysis of differentially expressed genes regulating antiviral immune response indicated that in contrast to RVA, RVC infection resulted in robust upregulation of expression of the genes encoding pattern recognition receptors including RIG1-like receptors and melanoma differentiation-associated gene-5. RVC infection was associated with a prominent upregulation of the most of glycosyltransferase-encoding genes except for the sialyltransferase-encoding genes which were downregulated similar to the effects observed for G9P[13]. CONCLUSIONS: Our results provide novel data highlighting the unique aspects of the RVC-associated host cellular signalling and suggest that increased upregulation of the key antiviral factors maybe one of the mechanisms responsible for RVC age-specific characteristics and its inability to replicate in most cell cultures.

Host Cell Response to Rotavirus Infection with Emphasis on Virus-Glycan Interactions, Cholesterol Metabolism, and Innate Immunity.

Although rotavirus A (RVA) is the primary cause of acute viral gastroenteritis in children and young animals, mechanisms of its replication and pathogenesis remain poorly understood. We previously demonstrated that the neuraminidase-mediated removal of terminal sialic acids (SAs) significantly enhanced RVA-G9P[13] replication, while inhibiting RVA-G5P[7] replication. In this study, we compared the transcriptome responses of porcine ileal enteroids (PIEs) to G5P[7] vs. G9P[13] infections, with emphasis on the genes associated with immune response, cholesterol metabolism, and host cell attachment. The analysis demonstrated that G9P[13] infection led to a robust modulation of gene expression (4093 significantly modulated genes vs. 488 genes modulated by G5P[7]) and a significant modulation of glycosyltransferase-encoding genes. The two strains differentially affected signaling pathways related to immune response, with G9P[13] mostly upregulating and G5P[7] inhibiting them. Both RVAs modulated the expression of genes encoding for cholesterol transporters. G9P[13], but not G5P[7], significantly affected the ceramide synthesis pathway known to affect both cholesterol and glycan metabolism. Thus, our results highlight the unique mechanisms regulating cellular response to infection caused by emerging/re-emerging and historical RVA strains relevant to RVA-receptor interactions, metabolic pathways, and immune signaling pathways that are critical in the design of effective control strategies.

Rotavirus C Replication in Porcine Intestinal Enteroids Reveals Roles for Cellular Cholesterol and Sialic Acids.

Rotaviruses (RVs) are a significant cause of severe diarrheal illness in infants and young animals, including pigs. Group C rotavirus (RVC) is an emerging pathogen increasingly reported in pigs and humans worldwide, and is currently recognized as the major cause of gastroenteritis in neonatal piglets that results in substantial economic losses to the pork industry. However, little is known about RVC pathogenesis due to the lack of a robust cell culture system, with the exception of the RVC Cowden strain. Here, we evaluated the permissiveness of porcine crypt-derived 3D and 2D intestinal enteroid (PIE) culture systems for RVC infection. Differentiated 3D and 2D PIEs were infected with porcine RVC (PRVC) Cowden G1P[1], PRVC104 G3P[18], and PRVC143 G6P[5] virulent strains, and the virus replication was measured by qRT-PCR. Our results demonstrated that all RVC strains replicated in 2D-PIEs poorly, while 3D-PIEs supported a higher level of replication, suggesting that RVC selectively infects terminally differentiated enterocytes, which were less abundant in the 2D vs. 3D PIE cultures. While cellular receptors for RVC are unknown, target cell surface carbohydrates, including histo-blood-group antigens (HBGAs) and sialic acids (SAs), are believed to play a role in cell attachment/entry. The evaluation of the selective binding of RVCs to different HBGAs revealed that PRVC Cowden G1P[1] replicated to the highest titers in the HBGA-A PIEs, while PRVC104 or PRVC143 achieved the highest titers in the HBGA-H PIEs. Further, contrasting outcomes were observed following sialidase treatment (resulting in terminal SA removal), which significantly enhanced Cowden and RVC143 replication, but inhibited the growth of PRVC104. These observations suggest that different RVC strains may recognize terminal (PRVC104) as well as internal (Cowden and RVC143) SAs on gangliosides. Finally, several cell culture additives, such as diethylaminoethyl (DEAE)-dextran, cholesterol, and bile extract, were tested to establish if they could enhance RVC replication. We observed that only DEAE-dextran significantly enhanced RVC attachment, but it had no effect on RVC replication. Additionally, the depletion of cellular cholesterol by MßCD inhibited Cowden replication, while the restoration of the cellular cholesterol partially reversed the MßCD effects. These results suggest that cellular cholesterol plays an important role in the replication of the PRVC strain tested. Overall, our study has established a novel robust and physiologically relevant system to investigate RVC pathogenesis. We also generated novel, experimentally derived evidence regarding the role of host glycans, DEAE, and cholesterol in RVC replication, which is critical for the development of control strategies.