Further experiments applying exogeneous soluble NA revealed that essentially the most most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA

onocytes had been isolated by gelatin adherence [27] and permitted to differentiate in RPMI (Life Technologies) supplemented with 20% fetal bovine serum (FBS), 500U/ml granulocyte-macrophage colony-stimulating issue (GM-CSF), and 250 U/ml recombinant human interleukin-4 (rIL-4) (each from Prospec-Tany, Israel). The medium was replaced each second day till day 6 to produce imDCs. P338D1 cells (American Tissue Culture Collection [ATCC] CCL-46), a macrophage-like cell line expressing Fcy-receptors, was maintained in DMEM (PAA Laboratories, Austria) supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.75% sodium bicarbonate (Invitrogen) and 1 mM sodium pyruvate (Gibco). Vero-WHO cells (European Collection of Cell Culture 88020401) have been maintained in DMEM supplemented with 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Human adenocarcinoma LoVo cells (ATCC CCL-229) have been maintained in ” Ham’s medium (Life Technologies) supplemented with 20% FBS. B cell lines Raji wild type (wt, ATCC CCL-86) and Raji DC-SIGN were maintained in RPMI (Life Technologies) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. The Raji DC-SIGN cell line was stably transfected having a plasmid coding for DC-SIGN [28]. All mammalian cells and cell lines where maintained at 37uC/5% CO2. C6/36 (ATCC CRL-1660), an Aedes albopictus cell line, was maintained in minimal necessary medium (Life Technologies) supplemented with 10% FBS, 25 mM HEPES, 7.5% sodium bicarbonate, 100 U/ml penicillin, one hundred mg/ ml streptomycin, 200 mM glutamine and one hundred mM nonessential amino acids at 30uC/5% CO2 imDCs were infected at a multiplicity of genome-containing particles (MOG) of 1000 of either immature DENV-2 or std DENV-2. At 1.5 hpi, fresh medium was added for the cells. Growth curve analysis showed that DENV-infected imDCs start off to secrete”
11033056” new particles at 24 hpi (information not shown). We decided to harvest at 43 hpi so we could measure the maximum output from the very first round of replication. The amount of developed infectious particles was measured by normal plaque assay on BHK-21 clone 15 cells. The detection limit on the plaque assay is 18 PFU/ ml [30]. The part of DC-SIGN was studied by incubating imDCs 1 h prior to and through infection with 25 mg/ml of either an antiDC-SIGN antibody or even a non-specific isotype ” handle (both R&D systems, MN, USA). To test if viral infectivity could be enhanced by antibodies, immature DENV-2 (MOG 1000) or, as a handle, std DENV-2 (MOG 100) was pre-opsonized with 10-fold sequential dilutions of human serum prior to infection. We used convalescent serum (28 days following infection) from a DENV-2 immune, hospitalized patient. For gain-of-function experiments, early passages from the stably transfected B cell line Raji DC-SIGN and as a manage, Raji wt had been infected with MOG 1000 of immature or std DENV-1, two and 4 under the same conditions as described above. Post-entry maturation of immature particles was blocked by treating cells with the furin inhibitor (FI) DecanoylRVKR-CMK (Calbiochem) prior (50 mM) and for the duration of (25 mM) virus infection. Infectivity assays on the macrophage-like cell line 28643-80-3 P388D1 have been performed under the same conditions as for imDCs. For antibody-dependent enhancement studies, P388D1 cells have been infected with human serum-opsonized immature DENV-1, two, and 4 at MOG 1000 or, as a manage, non-opsonized std DENV-1, two, and four at MOG 1000. For experiments in P388D1 and Raji cells, the amount of infectious particl