Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production

Epidemiologic, Experimental, and Clinical Links between Respiratory Syncytial Virus Infection and Asthma

Virtually all children experience respiratory syncytial virus (RSV) infection at least once during the first 2 years of life, but only a few develop bronchiolitis and more severe disease requiring hospitalization, usually in the first 6 months of life. Children who recover from RSV-induced bronchiolitis are at increased risk for the development of recurrent wheeze and asthma in later childhood. Recent studies suggest that there is an association between RSV-induced bronchiolitis and asthma within the first decade of life but that this association is not significant after age 13. Despite the considerable progress made in our understanding of several aspects of respiratory viral infections, further work needs to be done to clarify the molecular mechanisms of early interactions between virus and host cell and the role of host gene products in the infection process. This review provides a critical appraisal of the literature in epidemiology and experimental research which links RSV infection to asthma. Studies to date demonstrate that there is a significant association between RSV infection and childhood asthma and that preventing severe primary RSV infections can decrease the risk of childhood asthma

Rapid Detection of Listeria monocytogenes in Food and Food Animals

The objective of this study was to stream- line the detection and identification of Listeria monocytogenes (LM) in livestock and foods. Hog tonsil scrapings, hog tissues collected at necropsy, ground pork, and turkey washes were inoculated into UVM-1 (10% w/v) and after incubation (3 days) transferred to UVM-2. After 2 days, samples were plated to Palcam agar and incubated (48 h, 37 o C). Characteristic LM colonies were verified with the multiplex Polymerase chain reaction assay, which targets the 16S rRNA gene of Listeria and the hlyA gene unique to the species monocytogenes. To determine when LM achieved detectable levels during enrichment, template DNA was extracted directly from UVM-2 and Palcam agar and screened by multiplex PCR. When screened directly from UVM-2 by PCR, 34% (65 of 190) of ground pork samples and 19% (11 of 60) of turkey washes were positive for LM. In contrast, by multiplex PCR screening of colonies from Palcam agar 39% (75 of 190) of ground pork and 29% (18 of 60) of turkey wash samples were positive. Future studies will focus on improved recovery from UVM-2 with immunomagnetic beads. LM positive isolates (n=33) of ground pork were serotyped and assigned to type 4 (72%) and type 1 (14%). Forteen percent of isolates were neither serotype 1 nor 4. For live hogs, out of 150 samples each of carcasses, tonsils, and ileocecal lymph nodes tested, LM was detected once from tonsils and nodes by multiplex PCR. In contrast, LM was found on ~30% of ground pork produced from that same day from packing plant</p

Comparison of a Multiplex and 5' Nuclease PCR Assays for the Rapid Detection of Pathogenic Yersinia enterocolitica in Swine and Pork Products

Bacteriological culture methods were compared with PCR based protocols (multiplex PCR and TaqMan assay) for the rapid detection of pathogenic Yersinia enterocolitica (YE) in market weight hogs and pork products. The prevalence of YE was compared in lairaged hogs (n=150) versus hogs transported directly to the farm (n=150). By bacteriological culture, YE was not detected in any of the hog tissues tested but was detected by multiplex PCR and TaqMan assay. We also screened ground pork and chitterlings for the presence of YE. By standard culture, YE was detected in chitterlings (8%). By multiplex PCR, YE was identified in ground pork (10%) and chitterlings (27%). TaqMan assay identified YE in ground pork (44%) and chitterlings (79%).</p

A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models.

Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections

Transfection of cells in a 3D-VEC model.

<p>Cells in the 3D-VEC model were transfected with either chitosan or Lipofectamine 2000 or CNs complexed with pEGFP. At 48 h post-transfection, the tissues were fixed in 10% formalin, paraffin-embedded, and immunostained using anti-GFP antibody and nuclear-stained with DAPI. H&E stained histological (formalin-fixed) cross-sections of VLC-FT are also shown. LP, lamina propria; EL, epithelial layers.</p

Safety studies in RVI model.

<p>Rabbits (n = 9) were divided into three groups and in each group (n = 3) we applied cream alone, cream mixed with chlipid nanocomplexes, or cream mixed with psicocktail chlipid nanocomplexes every day for 9 days. In the RVI model, the CVL was collected at days 11 and 20, the cells were cytospun, fixed with 4% paraformaldehyde and permeabilized using 100% methanol. The cells were later treated with anti-CD45 tagged with Alexa Fluor 555 and viewed under a fluorescence microscope. Images were taken at 100x magnification. The two panels on the right show H&E-staining of the cells from CVL of rabbits treated as described above.</p

Blood chemistry of control and nanocomplexes treated monkeys.

<p>Blood chemistry of control and nanocomplexes treated monkeys.</p

Inhibition of HIV-1 replication by siRNAs using 3D VEC model.

<p>The 3-D VEC tissues were co-transfected with cocktail plasmid siRNAs-chlipid nanocomplexes or pU6 vector control-chlipid nanocomplexes or chlipid complexes alone with no plasmids. A p24 ELISA was performed on supernatants collected on days 0, 2, and 3 post-infection and p24 concentrations (pg/ml) were measured using p24-ELISA. Assays were run in duplicate on supernatants collected and expressed as mean p24 concentration in pg/ml ± SEM.</p