Role of zinc metalloprotease (ZMPSTE24) in porcine reproductive and respiratory syndrome virus (PRRSV) replication in vitro.

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 postinfection (hpi) between cells transfected with ZMPSTE24-FLAG and the vector control. Our results suggest that ZMPSTE24 overexpression may restrict PRRSV replication at a post-entry step.

Characterization of bovine ileal epithelial cell line for lectin binding, susceptibility to enteric pathogens, and TLR mediated immune responses.

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.

A chimeric influenza hemagglutinin delivered by parainfluenza virus 5 vector induces broadly protective immunity against genetically divergent influenza a H1 viruses in swine.

Pigs are an important reservoir for human influenza viruses, and influenza causes significant economic loss to the swine industry. As demonstrated during the 2009 H1N1 pandemic, control of swine influenza virus infection is a critical step toward blocking emergence of human influenza virus. An effective vaccine that can induce broadly protective immunity against heterologous influenza virus strains is critically needed. In our previous studies [McCormick et al., 2015; PLoS One, 10(6):e0127649], we used molecular breeding (DNA shuffling) strategies to increase the breadth of the variable and conserved epitopes expressed within a single influenza A virus chimeric hemagglutinin (HA) protein. Chimeric HAs were constructed using parental HAs from the 2009 pandemic virus and swine influenza viruses that had a history of zoonotic transmission to humans. In the current study, we used parainfluenza virus 5 (PIV-5) as a vector to express one of these chimeric HA antigens, HA-113. Recombinant PIV-5 expressing HA-113 (PIV5-113) were rescued, and immunogenicity and protective efficacy were tested in both mouse and pig models. The results showed that PIV5-113 can protect mice and pigs against challenge with viruses expressing parental HAs. The protective immunity was extended against other genetically diversified influenza H1-expressing viruses. Our work demonstrates that PIV5-based influenza vaccines are efficacious as vaccines for pigs. The PIV5 vaccine vector and chimeric HA-113 antigen are discussed in the context of the development of universal influenza vaccines and the potential contribution of PIV5-113 as a candidate universal vaccine.

Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf.

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.