Fig 1.
Growth properties of the two Taiwan H5N2 duck viruses in MDCK and DF1 cells.
Levels of M-vRNA in the supernatant of (a) MDCK and (b) DF1 cells infected with DV518 and DV413 at MOI of 0.01 and 0.1, respectively, were measured by quantitative RT-PCR. In addition, the viral replication capabilities of DV518 and DV413 in MDCK and DF1 cells at MOI of 0.1 were evaluated by the TCID50 method (c and e) and the hemagglutination assay (d and f). Supernatant samples from the infected cells were harvested at the indicated time points. The results are from two independent experiments, presented as means ± standard deviations of triplicate samples. Abbreviation: MOI: multiplicity of infection; TCID50: 50% tissue culture infectious dose.*p<0.05
Fig 2.
The distributions of plaque sizes in MDCK cells infected with DV518 and DV413 virus strains and the three plaque-purified virus strains.
Plaque sizes (mm2) and numbers of plaques were analyzed by software Image J 1.45A in a representative well infected by DV518 and DV413 virus strains and their plaque-purified strains (p518-L, p518-S, and p413). Mean is shown by a parallel bar, and the total number of plaques is shown at the top, marked as n = 56, 67, 12, 9, and 17, respectively.
Fig 3.
The growth kinetics of the three plaque-purified virus strains (p518-L, p518-S and p413) in MDCK and DF1 cells.
MDCK and DF1 cells were infected with p518-L, p518-S, and p413 virus strains at an MOI of 0.01. The supernatants were harvested at the indicated time points. The hemagglutination assays were used to examine the viral growths. The results are shown as means ± standard deviations of triplicate samples. MOI: multiplicity of infection
Fig 4.
Viral yield in chicken embryonated eggs inoculated with p518-L, p518-S, and p413 virus strains.
Five SPF eggs were inoculated with 4x105 TCID50 titer of each of the p518-L, p518-S, and p413 virus strains. Eggs were euthanized at 48 h p.i. and their allantoic fluid was collected for measurement of viral load by either TCID50 method (empty circles "ο”) or hemagglutination assay (solid circles “•”). Mean values are shown by parallel bars. Both p518-S and p518-L inoculated groups had one uninfected egg (hemagglutination titer <1), which was excluded from the analyses. SPF: Specific pathogen free. TCID50: 50% tissue culture infectious dose.
Table 1.
Amino acid changes detected in eight gene segments of the DV518 and DV413 strains.
Fig 5.
Cell binding ability of p518-L, p518-S and p413 virus strains to MDCK and DF1 cells.
MDCK and DF1 cell monolayers were infected with p518-L, p518-S and p413 viruses at the same copy number and incubated at 4°C for 1 h. After extensive wash, viral RNAs were extracted from the infected cells and influenza viral M RNAs were quantified by quantitative RT-PCR. The copy numbers of M RNA attached to the cells were normalized to that of DV30 in each set of data. Results are shown as means with standard deviations in three experiments. *p<0.05. RT-PCR: Reverse transcription-polymerase chain reaction.
Fig 6.
The viral polymerase activity of p518-S and p518-L strains in MDCK cells measured by minigenome assays.
The viral polymerase activity of p518-S and p518-L strains was measured by minigenome assays as described in Materials and Methods. Data are presented as the ratios of firefly luciferase signal to Renilla luciferase signal, with means and standard deviations in triplicate experiments.
Fig 7.
Evaluation of viral entry efficiency for p518-S and p518-L virus strains in 293T cells.
The 293T cells were infected with p518-S and p518-L viruses at an MOI of 2. Cell lysates were collected at 0.5, 1.5 and 6 h p.i. and were examined by Western blot using anti-NP and anti-actin antibodies. Levels of protein and relative ratios of NP/Actin were analyzed by software Image J. MOI: multiplicity of infection