Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution.lipid raft | Raman imaging | alkyne tag | supported monolayer | sphingomyelin

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Porcine parvoviruses are small non-enveloped DNA viruses, very resistant to inactivation, and ubiquitous in the global pig population. Porcine parvovirus type 1 (PPV1) has been known since the 1960s and is a major causative agent of reproductive failure in breeding herds. During the last decade, several new parvoviruses have been identified in pigs by molecular methods and have been consecutively designated as PPV2 through PPV6. Epidemiology data for these viruses are limited, and the impact of these newly recognized parvoviruses on pigs is largely unknown. To further generate knowledge on the distribution of PPVs in pigs, a total of 247 serum samples were collected from six commercial Polish pig farms during 2013-2015 and tested by PCR assays and ELISAs. The pigs ranged from two to 18 weeks of age at sample collection. Breeding herds supplying the investigated farms were routinely vaccinated against PPV1. While all growing pig samples were negative for PPV1 DNA, young pigs were frequently negative for PPV1 antibodies and seroconversion to PPV1 was commonly seen at 9-10 weeks of age. The PPV2 antibody detection was highest in young pigs (2-6-week-old) and decreased in older pigs indicating passively acquired antibodies. The DNA prevalence rates in the serum samples analysed were 19% for PPV2, 7.7% for PPV3, 2.4% for PPV4, 4.0% for PPV5 and 6.1% for PPV6. Most PPV DNA-positive samples were identified in 9- to 18-week-old pigs with no obvious association with disease on the farm. All recently emerging PPV genotypes were detected in Polish farms. Similar to previous reports in other pig populations, PPV2 was the most frequent PPV genotype circulating in Poland.