Figure 1.
Influenza viruses can spread independent of the NA activity.
(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 [29]. 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.
Figure 2.
NA-deficient influenza virus spreads through cell-to-cell transmission.
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.
Figure 3.
The cell-to-cell transmission of the NA-deficient influenza virus is less sensitive to the neutralizing antibody.
(A) Infection of the wild-type and (B) NA-deficient influenza virus were performed in the presence or absence of antiserum containing neutralizing antibodies. Immunofluorescence analyses were performed with cells infected with wild-type influenza virus at 18 hpi using anti-NP antibody and anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Invitrogen). GFP fluorescence derived from the recombinant virus was observed at 36 hpi. Scale bar, 100 µm. (C) The level of viral spreading was indicated in the graph by measuring NP and GFP derived from wild-type and NA-deficient virus, respectively. Five different microscope fields were taken randomly, and then the intensity of green color was analyzed with ImageJ NIH image processing software. Each result was represented by a value relative to that in the absence of neutralizing antibodies. Error bars indicate s.d. from 3 independent experiments.
Figure 4.
The cell-to-cell transmission of the NA-deficient influenza virus requires functional HA.
(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).
Figure 5.
The cell-to-cell transmission of the NA-deficient influenza virus occurs the apical cell surface.
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.
Figure 6.
Influenza viruses can not re-infect previously infected cells.
(A) A method for determination of the amount of segment 3 genome derived from ts53 and wild-type. Total RNA was reverse-transcribed with the primer PA-895-rev, which is complementary to the segment 3 positive-sense RNA. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut partially corresponding to segment 3 positive sense RNA between the nucleotide sequence positions 678 to 700 except for 696 and 697, which are shown in red letters. Since segment 3 of ts53 has a substitution mutation from U to C at the nucleotide position of 701, the PCR product derived from wild-type could be digested by Stu I but not that from ts53. Then, PCR products were digested with Stu I and separated through 8% PAGE. (B) Detection of the genome of the segment 3 derived from ts53 or wild-type. At 3 hours post superinfection of wild-type virus, total RNA was extracted, and semi-quantitative RT-PCR was performed. Subsequently, the amplified DNA products were digested with Stu I and separated through 8% PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively. The relative amount of wild-type segment 3 to that at 0 hour in the absence of oseltamivir phosphate was shown in the graph. Error bars indicate S.D. from 3 independent experiments. White bar, in the absence of oseltamivir phosphate; black bar, in the presence of oseltamivir phosphate.