The intestinal epithelium is a major site of interaction with pathogens. In bovine intestinal epithelial cells (BIECs), Toll-like receptors (TLRs) play an important role in innate immune responses against enteric pathogens. This study is aimed at establishing a stable bovine intestinal epithelial cell line that can be maintained by a continuous passage so that studies on innate immune responses against various enteric pathogens can be performed. The main goal was to establish pure cultures of primary and immortalized bovine intestinal epithelial cells from the ileum and then characterize them biochemically and immunologically. Mixed epithelial and fibroblast bovine ileal intestinal cultures were first established from a 2-day old calf. Limiting dilution method was used to obtain a clone of epithelial cells which was characterized using immunocytochemistry (ICC). The selected clone BIEC-c4 was cytokeratin positive and expressed low levels of vimentin, confirming the epithelial cell phenotype. Early passage BIEC-c4 cells were transfected with either simian virus 40 (SV40) large T antigen or human telomerase reverse transcriptase (hTERT), or human papillomavirus (HPV) type 16E6/E7 genes to establish three immortalized BIEC cell lines. The expression of SV40, hTERT and HPV E6/E7 genes in immortalized BIECs was confirmed by a polymerase chain reaction (PCR). Immunocytochemistry and immunofluorescence assays also confirmed the expression of SV40, hTERT and HPV E6 proteins. The immortalized BIECs were cytokeratin positive and all except HPV-BIECs expressed low levels of vimentin. A growth kinetics study indicated that there were no significant differences in the doubling time of immortalized BIECs as compared to early passage BIEC-c4 cells. All four BIEC types expressed TLR 1-10 genes, with TLR 3 and 4 showing higher expression across all cell types. These newly established early passage and immortalized BIEC cell lines should serve as a good model for studying infectivity, pathogenesis and innate immune responses against enteric pathogens.

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Highlights An HA-based vaccine candidate, created by DNA shuffling (HA-113), can be immunogenic when the recombinant antigen is expressed by PIV5 (PIV5-113). Immunity induced by the PIV5-113 vaccine can protect mice against infection with 4 of 5 parental HAs used to create the vaccine. Immunity induced by PIV5-113 can protect pigs against infection with an influenza virus isolate that is known to be infectious in pigs.

The transmembrane zinc metalloprotease, ZMPSTE24, works in cooperation with interferon induced transmembrane protein 3 (IFITM3) to restrict entry of several enveloped viruses. We investigated the role of ZMPSTE24 in porcine reproductive and respiratory syndrome virus (PRRSV) replication. ZMPSTE24 overexpression significantly reduced PRRSV replication in MARC-145 cells.Interestingly, knockdown of endogenous ZMPSTE24 did not significantly impact virus replication. There was no significant difference in the percentage of PRRSV positive cells and viral RNA copies at 3 hours post infection (hpi) between cells transfected with ZMPSTE24-FLAG and vector control. Our results suggest that ZMPSTE24 overexpression may restrict PRRSV replication at a post-entry step.

The transmembrane zinc metalloprotease, ZMPSTE24, works in cooperation with interferon induced transmembrane protein 3 (IFITM3) to restrict entry of several enveloped viruses. We investigated the role of ZMPSTE24 in porcine reproductive and respiratory syndrome virus (PRRSV) replication. ZMPSTE24 overexpression significantly reduced PRRSV replication in MARC-145 cells. Interestingly, knockdown of endogenous ZMPSTE24 did not significantly impact virus replication. There was no significant difference in the percentage of PRRSV positive cells and viral RNA copies at 3 hours post infection (hpi) between cells transfected with ZMPSTE24-FLAG and vector control. Our results suggest that ZMPSTE24 overexpression may restrict PRRSV replication at a post-entry step.