Table 1.
Amino acids at PB2 positions 627 and 701 of viruses used in this study.
Figure 1.
Plaque phenotypes of wild-type, PB2 627E and PB2 627E 701N viruses at 33°C and 37°C.
Plaque assays were performed on MDCK cells incubated at 33°C or 37°C, as indicated at the left of the figure, and immunostained with virus-specific antibody. (A) Plaque phenotypes of rPan99 WT and PB2 mutant viruses. (B) Average plaque diameter for each rPan99 virus is plotted at 33°C and 37°C. (C) Plaque phenotypes of rVN1203 WT and PB2 mutant viruses. (D) Average plaque diameter for each rVN1203 virus is plotted at 33°C and 37°C. Plaque assays of rPan99 viruses and rVN1203 viruses were performed at different times. Error bars indicate standard deviation. * indicates p<0.01 and ** indicates p<0.001, as determined by t-test of plaque diameter values.
Figure 2.
Growth kinetics of wild-type, PB2 627E and PB2 627E 701N viruses in MDCK cells incubated at 37°C and 33°C.
(A) and (B) show the results from rPan99-based viruses, while (C) and (D) show the results from rVN1203-based viruses. Titers of the wild-type viruses are shown with squares and solid lines; titers of PB2 627E mutants are shown with triangles and long dashed lines; and titers of PB2 627E 701N viruses are shown with circles and short dashed lines. The MOI of infection in all cases was 0.01 PFU/cell. ** all three viruses are significantly different from each other (p<0.05, Student's t-test). * the titers of both mutants are significantly different from those of the wild-type (p<0.05, Student's t-test). # the titers of the 627E mutant are significantly different from those of both the wild-type and the 627E 701N viruses. † the titers of the 627E 701N double mutant are significantly different from those of both the wild-type and the 627E single mutant viruses.
Figure 3.
Growth kinetics of rPan99 wild-type, rPan99 627E and rPan99 627E 701N viruses in the upper and lower respiratory tracts of guinea pigs.
(A) Viral titers in nasal wash samples of infected guinea pigs are plotted on days 2 and 4 p.i. On day 2 p.i., titers of the wild-type virus were significantly different from rPan99 627E (p = 0.0020) and from rPan99 627E 701N (p = 0.0057). On day 4 p.i., titers of the wild-type virus were not significantly different from those of either PB2 mutant virus (p≥0.03). (B) Titers of virus recovered from the supernatant of homogenized lung tissue at 2 d and 4 d p.i. Virus in three tissue samples from each of two animals was quantified; titers from one guinea pig are shown with open squares and titers from the second guinea pig are shown with closed diamonds. Titers of the wild-type virus were not significantly different from rPan99 627E (p = 0.39 on day 2 and p = 0.011 on day 4) or from rPan99 627E 701N (p = 0.067 on day 2 and p = 0.025 on day 4). All p-values were determined by Student's t-test. * indicates a significant difference relative to the wild-type, with p<0.01 as determined by Student's t-test. The mean and standard deviation for each data set are shown above the corresponding data points.
Figure 4.
Aerosol transmission of rPan99 wild-type, rPan99 627E and rPan99 627E 701N viruses in guinea pigs.
Nasal wash titers are plotted as a function of time post-inoculation. Titers of intranasally inoculated animals are represented by dashed lines and filled squares; titers of exposed guinea pigs are shown with solid lines and filled triangles. (A) The cumulative results of two experiments with rPan99 wild-type virus are shown. All four exposed guinea pigs were infected in both experiments. (B) The cumulative results of three experiments with rPan99 627E are shown. One of four, three of four and two of four exposed guinea pigs became infected in the first, second and third experiments, respectively. (C) The cumulative data from three experiments with rPan99 627E 701N are shown. Two of four, four of four, and four of four exposed guinea pigs became infected in the first, second, and third experiments, respectively.
Table 2.
Virus detection and seroconversion in guinea pigs inoculated or exposed as part of rPan99 virus transmission experiments.
Figure 5.
Contact transmission of rVN1203 wild-type, rVN1203 627E and rVN1203 627E 701N viruses in guinea pigs.
Nasal wash titers are plotted as a function of time post-inoculation. Titers of intranasally inoculated animals are represented by dashed lines and filled squares; titers of exposed guinea pigs are shown with solid lines and filled triangles. (A) Transmission of rVN1203 wild-type virus. (B) Transmission of the rVN1203 627E mutant. (C) Transmission of the rVN1203 627E 701N double mutant.
Table 3.
Guinea pig 50% infectious dose of wild-type and PB2 mutant viruses.