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Porcine epidemic diarrhea virus (PEDV) frequently causes diarrhea outbreaks. However, whether newly discovered enteric viruses such as porcine kobuvirus (PKV) and porcine astroviruses (PAstVs) are also correlated with diarrhea is still unclear. Diarrhea outbreaks were reported in a PEDV-vaccinated pig farm in Xinjiang Uygur Autonomous Region of China from 2019 to 2020. PEDV was a common pathogen detected in fecal samples by routine RT-PCR assays. The PEDV positive fecal sample was used for pathogenic analysis due to the failure isolation of PEDV. The challenged neonatal piglets appeared watery diarrhea within one day post infection (dpi) and all died within 6 dpi. Histopathological and immunohistochemical examinations supported that PEDV is a major pathogen causing intestinal lesions. To further explore enteric viruses associated with neonatal piglet diarrhea, metagenomics sequencing was performed for the diarrheic piglets. Remarkably, PKV was the most abundant virus (58.33%) followed by PEDV (34.45%) and PAstVs (7.22%), which were also confirmed by real-time RT-PCR assays. Significant in vivo replications of PEDV and PKV could only be observed in challenged piglets whilst PAstVs maintained similar virus loads in both challenged and mock infected piglets. Overall, this study provides first pathogenic and metagenomic evidence that significant proliferations of PEDV and PKV are closely associated with severe diarrhea in neonatal piglets, while PAstVs likely play limited roles in neonatal piglet diarrhea.

Porcine reproductive and respiratory syndrome virus (PRRSV) isolates show high genetic and pathogenic diversity. The mechanisms underlying different virulence of PRRSV isolates are still not fully clarified. Two highly homologous PRRSV isolates (XJ17-5 and JSTZ1712-12) with distinct virulence were identified in our previous study. To evaluate the association between host responses and different virulence, here we investigated the transcriptomic profiles of porcine alveolar macrophages (PAMs) infected with these two isolates. RNA-Seq results showed that there are 1932 differential expression genes (DEGs) between two PRRSV infected groups containing 1067 upregulation and 865 downregulation genes. Compared with the avirulent JSTZ1712-12 infected group, GO analysis identified significant enrichment gene sets not only associated with virus infection but also innate immune response in the virulent XJ17-5 infected group. In addition, KEGG analysis indicated significantly enriched genes associated with NOD-like and RIG-I-like receptor signaling pathways in XJ17-5 vs JSTZ1712-12 group. Furthermore, XJ17-5 isolate induced significantly higher levels of innate immune response associated genes (IL-1β, CXCL2, S100A8, OAS2, MX1, IFITM3, ISG15 and IFI6) than JSTZ1712-12 isolate, which were further confirmed by real-time PCR. Given that these two isolates share similar replication efficiency in vivo and in vitro, our results indicated that distinct virulence of PRRSV isolates is associated with different host innate immune responses.