Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 6

Figure 6. Respiratory droplet transmissibility of rNY1682-WT and rNY1682-D701N viruses in ferrets.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

Three ferrets were inoculated with 106 PFU of (A) rNY1682-WT or (B) rNY1682-D701N virus. All ferrets were housed individually in specialized cages that permit exchange of respiratory droplets, but prevent direct or indirect contact between inoculated-contact animal pairs. A naïve ferret was placed in an adjacent cage to an inoculated ferret 1 day post-inoculation (0 day post-contact). Viral replication was determined by titration of nasal washes collected on alternate days from inoculated (left bars) and contact (right bars) ferrets. Values for individual ferrets are shown and the dotted lines at 1 log10 indicate the lower limit of detection of infectious virus.

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.
2.
Figure 4

Figure 4. Analysis of viral replication efficiency in the respiratory tracts of mice.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

Six-week-old female BALB/cJ mice (n=3/group/time-point) were inoculated intranasally with 50 µl containing 103 TCID50 of rNY1682-WT (WT) or rNY1682-D701N (D701N). Animals were euthanized at 12, 24, 48, 96 hpi. The entire lung of each animal was homogenized in 1 ml of media and clarified by centrifugation, and nasal washes were collected from each mouse in 1 ml of media. (A) Viral titers of clarified lung homogenates or (B) nasal washes were determined by TCID50 assay using MDCK cells. The average of each group is shown with error bars representing SD(+/-), and * indicates a statistical difference between WT and D701N (p<0.05, ANOVA). The dotted lines at 1.5 log10 indicate the lower limit of detection of infectious virus.

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.
3.
Figure 2

Figure 2. Replication kinetics of rNY1682-WT and rNY1682-D701N in vitro.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

(A) Confluent monolayers of Madin-Darby canine kidney (MDCK) cells were inoculated with the rNY1682-WT (WT) or rNY1682-D701N (D701N) viruses at 0.01 TCID50/cell. Supernatants were collected at 2, 24, 48 and 72 hours post inoculation (hpi). (B) Confluent mouse rectal epithelial carcinoma cells (CMT-93) were inoculated with rNY1682-WT (WT) or rNY1682-D701N (D701N) viruses at 0.01 TCID50/cell and supernatants were harvested at 12, 24, 48, 72 and 96 hpi. (C, D, E) Confluent human lung epithelial cells (Calu-3) were inoculated with rNY1682-WT (WT) or rNY1682-D701N (D701N) viruses at 0.02 TCID50/cell and supernatants were harvested at 2, 24, 48, 72 and 96 hpi. Cells were incubated at 33oC (C), 37oC (D), or 39oC (E) for the respective growth curves. The averages of triplicate experiments are shown with error bars (+/-) representing the standard deviation, and * indicates that the differences were statistically significant (p<0.001, ANOVA).

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.
4.
Figure 1

Figure 1. PB2-D701N substitution enhances viral RNA polymerase activity in human cells.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

HEK-293T cells were transfected with pPolI-NS-Luc plasmid (pBZ81A36) that expresses negative sense virus-like RNA encoding a destabilized firefly luciferase enzyme that can be transcribed by the viral RdRp. The HEK-293T cells were also co-transfected with plasmids expressing the NY1682 PB1, PA and NP, and one of the PB2 clones (WT, or D701N) to generate different viral RdRp. Cells were also co-transfected with a Renilla luciferase expression plasmid to control for transfection efficiency. After 6 h and 18 h incubation at 33oC, 37oC, or 39oC, both firefly and Renilla luciferase production were measured, and Renilla expression was used to normalize the data. The averages of triplicate experiments are shown with error bars that represent standard deviation (SD). * Indicates that the difference between the PB2-WT and PB2-D701N was statistically significant (p<0.05, t-test).

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.
5.
Figure 5

Figure 5. Pathogenicity of rNY1682-WT, rNY1682-E158G, rNY1682-E627K, and rNY1682-D701N viruses in mice.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

Six-week-old female BALB/cJ mice (n=5/group) were inoculated intranasally with 50 µl containing 105 TCID50 of different recombinant viruses (rNY1682-WT (WT), rNY1682-E158G (E158G), rNY1682-E627K (E627K), or rNY1682-D701N (D701N)) or they were mock inoculated (Mock). (A) Morbidity was assessed by weight loss over a 14 day period and is graphed as a percentage of the animals weights on the day of inoculation (day 0). The average body weight of each group is shown with error bars representing SD (+/-), and a significant difference between D701N and WT or E627K was evident as early as 4 d.p.i (*, p<0.01, ANOVA). (B) Mortality associated with infection by the recombinant viruses (rNY1682-WT (WT) or rNY1682-D701N (D701N)) was also examined. Five-week-old female BALB/cJ mice (n=5/group) were inoculated intranasally with 50 µl containing 1×103, 104, 105, or 106 TCID50 of rNY1682-D701N or they were mock inoculated. (C) Morbidity was assessed by weight changes over a 14 day period and it is graphed as a percentage of the weight on the day of inoculation (day 0). The average body weight of each group is shown with error bars representing SD. (D) Mortality of the mice was also monitored for 14 days post inoculation. In accordance with our animal welfare protocol, mice that lost more than 25% of their original weight were euthanized for humane reasons.

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.
6.
Figure 3

Figure 3. Analysis of cytokine response and virus replication in primary human alveolar epithelial cells.. From: Asparagine Substitution at PB2 Residue 701 Enhances the Replication, Pathogenicity, and Transmission of the 2009 Pandemic H1N1 Influenza A Virus.

Human alveolar type I-like cells were infected at 37°C with MOI of 0.01 PFU/cell of the rNY1682-WT (WT) or rNY1682-D701N (D701N) virus, or were mock infected (Mock). Supernatants from the culture were collected at various time points post infection. (A) Supernatants collected at 1, 12, 24, 48, and 72 hpi were determined for viral titers by plaque assay. Representative growth kinetics of the WT and D701N viruses in one donor cells are shown. Supernatants collected at 24 hpi were analyzed by ELISA for the levels of (B) IFN-λ, (C) CCL5, (D) IL-6, and (E) IL-8. Each symbol represents an average value of 2 or 3 repeats using the same cells from one donor and thus seven different symbols represent the data from seven independent experiments performed using cells from seven donors. Due to the variation in cytokine responses among different donors, for each donor, cytokine/chemokine induced by WT was set as 100% and cytokine/chemokine induced by D701N or Mock was presented as a percentage of WT. Horizontal bar in each group indicates the average value from seven donors and * indicates a statistical difference between WT and D701N (p<0.05, ANOVA).

Bin Zhou, et al. PLoS One. 2013;8(6):e67616.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk