Human rotavirus (HRV) is a major cause of viral gastroenteritis in infants; particularly in developing countries where malnutrition is prevalent. Malnutrition perturbs the infant gut microbiota leading to sub-optimal functioning of the immune system and further predisposing infants to enteric infections. Therefore, we hypothesized that malnutrition exacerbates rotavirus disease severity in infants. In the present study, we used a neonatal germ free (GF) piglets transplanted with a two-month-old human infant’s fecal microbiota (HIFM) on protein deficient and sufficient diets. We report the effects of malnourishment on the HRV infection and the HIFM pig microbiota in feces, intestinal and systemic tissues, using MiSeq 16S gene sequencing (V4-V5 region). Microbiota analysis indicated that the HIFM transplantation resulted in a microbial composition in pigs similar to that of the original infant feces. This model was then used to understand the interconnections between microbiota diversity, diet, and HRV infection. Post HRV infection, HIFM pigs on the deficient diet had lower body weights, developed more severe diarrhea and increased virus shedding compared to HIFM pigs on sufficient diet. However, HRV induced diarrhea and shedding was more pronounced in non-colonized GF pigs compared to HIFM pigs on either sufficient or deficient diet, suggesting that the microbiota alone moderated HRV infection. HRV infected pigs on sufficient diet showed increased microbiota diversity in intestinal tissues; whereas, greater diversity was observed in systemic tissues of HRV infected pigs fed with deficient diet. These results suggest that proper nourishment improves the microbiota quality in the intestines, alleviates HRV disease and lower probability of systemic translocation of potential opportunistic pathogens/pathobionts. In conclusion, our findings further support the role for microbiota and proper nutrition in limiting enteric diseases.

Porcine deltacoronavirus (PDCoV) is an emerging infectious disease of swine with zoonotic potential. Phylogenetic analysis suggests that PDCoV originated recently from a host-switching event between birds and mammals. Little is known about how PDCoV interacts with its differing hosts. Human-derived cell lines are susceptible to PDCoV infection. Herein, we compare the gene expression profiles of an established host swine cells to potential emerging host human cells after infection with PDCoV. Cell lines derived from intestinal lineages were used to reproduce the primary sites of viral infection in the host. Porcine intestinal epithelial cells (IPEC-J2) and human intestinal epithelial cells (HIEC) were infected with PDCoV. RNA-sequencing was performed on total RNA extracted from infected cells. Human cells exhibited a more pronounced response to PDCoV infection in comparison to porcine cells with more differentially expressed genes (DEGs) in human, 7486, in comparison to pig cells, 1134. On the transcriptional level, the adoptive host human cells exhibited more DEGs in response to PDCoV infection in comparison to the primary pig host cells, where different types of cytokines can control PDCoV replication and virus production. Key immune-associated DEGs and signaling pathways are shared between human and pig cells during PDCoV infection. These included genes related to the NF-kappa-B transcription factor family, the interferon (IFN) family, the protein-kinase family, and signaling pathways such as the apoptosis signaling pathway, JAK-STAT signaling pathway, inflammation/cytokine–cytokine receptor signaling pathway. MAP4K4 was unique in up-regulated DEGs in humans in the apoptosis signaling pathway. While similarities exist between human and pig cells in many pathways, our research suggests that the adaptation of PDCoV to the porcine host required the ability to down-regulate many response pathways including the interferon pathway. Our findings provide an important foundation that contributes to an understanding of the mechanisms of PDCoV infection across different hosts. To our knowledge, this is the first report of transcriptome analysis of human cells infected by PDCoV.

Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and their gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-S_HKU17, and icPDCoV-RBD_ISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-S_HKU17- and icPDCoV-RBD_ISU virus infections. In 5-day-old ECEs, significant mortality was observed in PDCoV inoculated eggs with less in the spike chimeras, while in 11-day-old ECEs there was no evidence of viral replication, suggesting that PDCoV is better adapted to cross species infection and differentiated ECE cells are not susceptible to PDCoV infection. Collectively, we demonstrate that the SpCoV chimeric viruses are not more infectious in turkeys, nor ECEs than wild type PDCoV. Therefore, understanding the cell and host factors that contribute to resistance to PDCoV and avian-swine chimeric virus infections may aid in the design of novel antiviral therapies against DCoVs.

We evaluated the effects of the probiotic Escherichia coli Nissle 1917 (EcN) and the antibiotic Ciprofloxacin (Cipro) on mRNA expression of intestinal epithelial cells (IEC) in gnotobiotic (Gn) piglets colonized with a defined commensal microflora (DMF) and inoculated with human rotavirus (HRV) that infects IECs. We analyzed mRNA levels of IEC genes for enteroendocrine cells [chromogranin A (CgA)], goblet cells [mucin 2 (MUC2)], transient amplifying progenitor cell [proliferating cell nuclear antigen (PCNA)], intestinal epithelial stem cell (SOX9) and enterocytes (villin). Cipro treatment enhanced HRV diarrhea and decreased the mRNA levels of MUC2 and villin but increased PCNA. These results suggest that Cipro alters the epithelial barrier, potentially decreasing the numbers of mature enterocytes (villin) and goblet cells secreting protective mucin (MUC2). These alterations may induce increased IEC proliferation (PCNA expression) to restore the integrity of the epithelial layer. Coincidental with decreased diarrhea severity in EcN treated groups, the expression of CgA and villin was increased, while SOX9 expression was decreased representing higher epithelial integrity indicative of inhibition of cellular proliferation. Thus, EcN protects the intestinal epithelium from damage by increasing the gene expression of enterocytes and enteroendocrine cells, maintaining the absorptive function and, consequently, decreasing the severity of diarrhea in HRV infection.