Prevalence and Antimicrobial Resistance of Salmonella in Market Raw Oysters in Taiwan.

We tested 137 samples of domestic shucked oysters and 114 samples of imported oysters collected from traditional retail markets and supermarkets during 2010 and 2011 in Taiwan for the presence of Salmonella. We obtained a total of 91 Salmonella isolates, representing nine serotypes, from 80 of the domestic samples. We did not find any Salmonella in the imported oysters. The presence of Salmonella contamination tended to be specific to the area from which the oysters were harvested: the Dongshih area had a significantly higher contamination rate (68.8%) than the Budai (20.0%) and Wanggong (9.1%) areas. In addition, the rate of Salmonella contamination was higher in oysters that were packed or sold with water (P < 0.05). The most commonly identified Salmonella serotypes were Saintpaul (26.4%), Newport (22.0%), and Infantis (13.2%). We screened the isolates for susceptibility to nine antimicrobials and compared them genetically by using PCR for the class 1 integron (int1), tetA, tetB, and blaPSE-1 genes. Eighteen isolates (19.8%) were resistant to at least one antimicrobial agent, and the most frequent resistances were those to tetracycline and oxytetracycline (n = 12, 14.3%).We detected the antimicrobial resistance genes int1, tetA, tetB, and blaPSE-1 in 16.5, 26.4, 6.6, and 22.0% of the isolates, respectively. Eleven of the 18 antimicrobial-resistant isolates contained one or two int1 cassettes, suggesting that the presence of int1 is highly correlated with antimicrobial resistance in Salmonella isolates from oysters. The consumption of oysters is increasing in Taiwan, and information related to Salmonella contamination in oysters is rather limited. Our results indicate that raw oyster consumption from retail markets in Taiwan is associated with a human health hazard owing to Salmonella, including multidrug-resistant Salmonella strains.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22.

UNLABELLED: Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE: Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

Establishing Restricted Expression of Caveolin-1 in HIV Infected Cells and Inhibition of Virus Replication.

BACKGROUND: Caveolin-1 (Cav-1) is the major protein of the caveolae and plays a role in multiple cellular functions and implicated to have anti-HIV activity. Regulated expression of Cav-1 is important for safe and effective use in order to exploit Cav-1 for HIV therapeutic applications. METHODS: A series of Cav-1 and GFP expression vectors were constructed under the control of the HIV LTR for conditional expression or CMV promoter and the expression of Cav-1 was monitored in the presence or absence of Tat or HIV infection in order to establish the restricted expression of Cav-1 to HIV infected cells. RESULTS: Cav-1 expression was evident under the control of the HIV LTR in the absence of Tat or HIV infection as demonstrated by immunoblot. Placing two internal ribosomal entry sequences (IRES) and a Rev response element, RRE (5'~ LTR-IRES-GFP-RRE-IRES-Cav-1~3') resulted in no expression of Cav-1 in the absence of Tat with effective expression in the presence of Tat. Transduction of HIV permissive cells with this construct using a foamy virus vector show that Cav-1 was able to inhibit HIV replication by 82%. Cells that received LTR-IRES-GFP-RRE-IRES-Cav-1 remain healthy in the absence of Tat or HIV infection. CONCLUSION: These results taken together reveal the inclusion of two IRES establishes a significant reduction of leak through expression of Cav-1 in the absence of Tat or HIV infection. Such regulated expression will have therapeutic application of Cav-1 for HIV infection as well as broad applications which can be beneficial for other host-targeted interventions as therapeutics.

Caveolin-1 modulates HIV-1 envelope-induced bystander apoptosis through gp41.

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4(+) T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4(+) T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.

The foamy virus genome remains unintegrated in the nuclei of G1/S phase-arrested cells, and integrase is critical for preintegration complex transport into the nucleus.

Foamy viruses are a member of the spumavirus subfamily of retroviruses with unique mechanisms of virus replication. Foamy virus replication is cell cycle dependent; however, the genome is found in the nuclei of cells arrested in the G(1)/S phase. Despite the presence of genome in the nuclei of growth-arrested cells, there is no viral gene expression, thus explaining its dependency on cell cycle. This report shows that the foamy virus genome remains unintegrated in G(1)/S phase-arrested cells. The foamy virus genome is detected by confocal microscopy in the nuclei of both dividing and growth-arrested cells. Alu PCR revealed foamy virus-specific DNA amplification from genomic DNA isolated in cycling cells at 24 h postinfection. In arrested cells no foamy virus DNA band was detected in cells harvested at 1 or 7 days after infection, and a very faint band that is significantly less than DNA amplified from cycling cells was observed at day 15. After these cells were arrested at the G(1)/S phase for 1, 7, or 15 days they were allowed to cycle, at which time foamy virus-specific DNA amplification was readily observed. Taken together, these results suggest that the foamy virus genome persists in nondividing cells without integrating. We have also established evidence for the first time that the foamy virus genome and Gag translocation into the nucleus are dependent on integrase in cycling cells, implicating the role of integrase in transport of the preintegration complex into the nucleus. Furthermore, despite the presence of a nuclear localization signal sequence in Gag, we observed no foamy virus Gag importation into the nucleus in the absence of integrase.