West Nile virus (WNV) is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

Background: Bluetongue virus (BTV), an arthropod-borne member of the Reoviridae family, is a double-stranded RNA segmented virus that causes an economically important livestock disease which has spread across Europe in recent decades. It can infect many species of domestic and wild ruminants including sheep, deer, cattle and goats. Type I interferon (alpha/beta interferon [IFN-␣/␤]) production was reported in vivo and in vitro upon BTV infection. However the cellular sensors and signaling pathways involved in this process remain unknown.Methods: The effect of BTV strains and replication on IFN-␤ production during a kinetic of infection was assessed at the mRNA level by real-time quantitative RT-PCR (RT-q-PCR) and at the protein level by IFN-␤ ELISA. The involvement of the IRF3 and NF-kB transcriptional factors in the IFN-␤ production was determined by immunoblot and immunofluorescence analyses. SiRNA-mediated knockdown of several pattern-recognition receptors (PRRs) was used to determine their contribution in the IFN-␤ production following BTV infection.Results: Upon BTV infection of A549 cells, expression of IFN-␤ and other pro-inflammatory cytokines was strongly induced at both protein and mRNA levels. This production appeared to be dependent on virus replication, since infection with UV-inactivated virus could no longer induce IFN-␤. We could also demonstrate that BTV infection activated the IRF3 and NF-kB pathways. Interestingly, the expression of IFN-␤ mRNA was greatly reduced after siRNA-mediated knockdown of the RNA helicases retinoic acidinducible gene-I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5), or their common adaptor protein mitochondrial antiviral signaling protein (MAVS). In contrast, silencing of MyD88, Toll-like receptor-3 (TLR-3) or the recently described DexD/H-box helicase DDX1 sensor had no effect on IFN-␤ mRNA induction. Finally, we found that overexpression of either RIG-I or MDA5 severely impaired BTV expression in infected A549 cells. Conclusion:These results suggest that the RIG-I-like (RLR) pathway is specifically engaged for IFN-␤ production following BTV infection and indicate that RIG-I and MDA5 can both contribute to its recognition and control.

Pasteurella multocida can infect a multitude of wild and domesticated animals, with infections in cattle resulting in hemorrhagic septicemia (HS) or contributing to bovine respiratory disease (BRD) complex. Current cattle vaccines against P. multocida consist of inactivated bacteria, which only offer limited and serogroup specific protection. Here, we describe a newly identified surface lipoprotein, PmSLP, that is present in nearly all annotated P. multocida strains isolated from cattle. Bovine associated variants span three of the four identified phylogenetic clusters, with PmSLP-1 and PmSLP-2 being restricted to BRD associated isolates and PmSLP-3 being restricted to isolates associated with HS. Recombinantly expressed, soluble PmSLP-1 (BRD-PmSLP) and PmSLP-3 (HS-PmSLP) vaccines were both able to provide full protection in a mouse sepsis model against the matched P. multocida strain, however no cross-protection and minimal serum IgG cross-reactivity was identified. Full protection against both challenge strains was achieved with a bivalent vaccine containing both BRD-PmSLP and HS-PmSLP, with serum IgG from immunized mice being highly reactive to both variants. Year-long stability studies with lyophilized antigen stored under various temperatures show no appreciable difference in biophysical properties or loss of efficacy in the mouse challenge model. PmSLP-1 and PmSLP-3 vaccines were each evaluated for immunogenicity in two independent cattle trials involving animals of different age ranges and breeds. In all four trials, vaccination with PmSLP resulted in an increase in antigen specific serum IgG over baseline. In a blinded cattle challenge study with a recently isolated HS strain, the matched HS-PmSLP vaccine showed strong efficacy (75–87.5% survival compared to 0% in the control group). Together, these data suggest that cattle vaccines composed of PmSLP antigens can be a practical and effective solution for preventing HS and BRD related P. multocida infections.

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