Journal: PLoS ONE

Article Title: Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

doi: 10.1371/journal.pone.0028178

Figure Lengend Snippet: (A) MDCK cells were infected with influenza virus A/WSN/33 at a multiplicity of infection (MOI) of 0.001 PFU per cell. At 48 hours post infection (hpi), culture supernatant was collected, and then its virus titer was determined by plaque assays. Each result was represented by a value relative to that in the absence of the drug. Error bars indicate standard deviation (s.d.) from 3 independent experiments. (B) Confluent MDCK cells were infected by wild-type influenza virus A/WSN/33 or NA-deficient influenza virus at MOI of 0.0001 in the presence or absence of 50 µg/ml oseltamivir phosphate. NA-deficient influenza virus was generated by reverse genetics as previously described . After incubation at 37°C for 36 hours, immunofluorescence analyses were performed using anti-nucleoprotein (NP) polyclonal antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 568 (Invitrogen). Scale bar, 100 µm.

Article Snippet: Confluent MDCK cell monolayer was prepared on transwell inserts (BD Falcon, pore size 0.4 μm) and infected with the NA-deficient influenza virus at MOI of 0.0001.

Techniques: Infection, Virus, Standard Deviation, Generated, Incubation, Immunofluorescence

Journal: PLoS ONE

Article Title: Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

doi: 10.1371/journal.pone.0028178

Figure Lengend Snippet: Confluent MDCK cells were infected with the NA-deficient influenza virus at MOI of 0.0001. After incubation at 37°C for 24 hours, a single GFP-positive cell, in which the recombinant virus replicated, was found at 1 hour after starting monitoring, and then this cell and its neighborhood were traced during the period from 24 hpi to 48 hpi at interval of 1 hour. Scale bar, 50 µm.

Article Snippet: Confluent MDCK cell monolayer was prepared on transwell inserts (BD Falcon, pore size 0.4 μm) and infected with the NA-deficient influenza virus at MOI of 0.0001.

Techniques: Infection, Virus, Incubation, Recombinant

Journal: PLoS ONE

Article Title: Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

doi: 10.1371/journal.pone.0028178

Figure Lengend Snippet: (A) Confluent MDCK cells were infected with the NA-deficient influenza virus at MOI of 0.0001 in the presence or absence of 1 µg/ml trypsin. GFP fluorescence derived from the recombinant virus was observed at 36 hpi. Scale bar, 100 µm. (B) MDCK cells were infected with influenza virus A/Udorn/72 at moi of 0.0001 in the presence or absence of 50 µM amantadine or 50 µg/ml oseltamivir phosphate. Amantadine at the concentration of 50 µM almost completely inhibited the production of progeny virions (data not shown). After incubation for 12, 24, and 48 h, immunofluorescence analyses were performed using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). Viral NP and nuclear DAPI staining are shown in green and blue, respectively. Scale bar, 100 µm. (C) Median sizes of clusters were shown as box plots summarizing sizes of 60 individual infectious foci formed in the presence of oseltamivir alone, or both oseltamivir and amantadine. Immunofluorescence analyses were performed as described in (B) at 24 hpi. Boxes enclose the lower and upper quartiles; thick horizontal lines represent the median; dashed lines indicate the extreme values; and black dots are outliers of individual infectious foci. The size of infectious foci was measured with AxioVision Release 4.7.2 imaging software (Carl Zeiss). Median sizes shown in red letters were clearly different from each other (p<0.01).

Article Snippet: Confluent MDCK cell monolayer was prepared on transwell inserts (BD Falcon, pore size 0.4 μm) and infected with the NA-deficient influenza virus at MOI of 0.0001.

Techniques: Infection, Virus, Fluorescence, Derivative Assay, Recombinant, Concentration Assay, Incubation, Immunofluorescence, Staining, Imaging, Software

Journal: PLoS ONE

Article Title: Tamiflu-Resistant but HA-Mediated Cell-to-Cell Transmission through Apical Membranes of Cell-Associated Influenza Viruses

doi: 10.1371/journal.pone.0028178

Figure Lengend Snippet: Confluent MDCK cells were prepared in transwell inserts and infected with the NA-deficient influenza virus at MOI of 0.0001 in the presence or absence of 0.3% (v/v) antiserum containing neutralizing antibodies (nAb) to influenza A virus. After virus adsorption, the antiserum was added from apical or basolateral side. GFP fluorescence derived from the recombinant virus was observed at 36 hpi. The antiserum added from the apical side could markedly block the cell-to-cell transmission of the NA-deficient influenza virus, whereas the antiserum added from the basolateral side could not. Scale bar, 100 µm.

Article Snippet: Confluent MDCK cell monolayer was prepared on transwell inserts (BD Falcon, pore size 0.4 μm) and infected with the NA-deficient influenza virus at MOI of 0.0001.

Techniques: Infection, Virus, Adsorption, Fluorescence, Derivative Assay, Recombinant, Blocking Assay, Transmission Assay