Figure 1.
Crystal structure of the A/Vietnam/1203/04 (H5N1) NA molecule (Protein Data Bank:2HTY).
Shown are 11 residues in or near the enzyme’s active site (black) that were substituted in this study. Residues shown in purple are positions where a single amino acid substitution affects the stability of the recombinant A/Turkey/15/06-like (H5N1) influenza virus.
Table 1.
Growth of Recombinant H5N1 Influenza Viruses Before and After Passaging in MDCK Cells.
Table 2.
Enzymatic Properties of the Neuraminidase of Recombinant H5N1 Influenza Viruses.
Figure 2.
NA enzyme kinetics of the recombinant A/Turkey/15/06-like (H5N1) viruses.
Substrate conversion velocity (V0) of NA is shown as a function of substrate concentration. Fluorogenic MUNANA substrate was used at a final concentration of 0 to 2000 µM. The viruses were standardized to an equivalent dose of 107.5 PFU/ml. Fluorescence was measured every 92 sec for 45 min at 37°C, using excitation and emission wavelengths of 355 and 460 nm, respectively.
Figure 3.
Patterns of clinical outcome in ferrets inoculated with the recombinant A/Turkey/15/06-like (H5N1) viruses.
(A) Mild, prolonged illness, (B) mild, brief illness, and (C) severe illness or (comparable to WT) caused by the indicated recombinant viruses. Shown are change in body temperature and weight, total number of inflammatory cells and protein concentrations in nasal washes, and virus titers in the upper respiratory tract. The horizontal lines show the mean inflammatory cell counts and protein concentrations in the nasal washes of uninoculated animals. Values are the mean ± s.d. for three ferrets. The mean s.d. of all data points for change in body temperature and weight was ∼±4.5%. *, P<0.05, **, P<0.01 compared to WT virus (one-way ANOVA).
Table 3.
Pathogenicity of the Recombinant H5N1 Influenza Viruses in Ferrets.
Figure 4.
Replication of recombinant WT, E119A, and N294S viruses in internal organs and histologic changes in the lungs morphology of ferrets infected with these H5N1 viruses.
(A) Virus titers were determined for the lungs (4 lobes tested separately), nasal turbinates, tracheas, small intestines, and livers of ferrets on day 4 post-inoculation. Values (log10EID50/gram tissue) are the mean ± s.d. for two ferrets, unless it is indicated that the virus was detected in one of two ferrets. *, P<0.05, **, P<0.01 compared to WT virus (one-way ANOVA). (B) The images shown are hematoxylin-and-eosin-stained sections of lung tissue from ferrets inoculated with the WT, E119A, and N294S viruses obtained on day 4 post-inoculation. Lung showing bronchiole (b) with epithelial necrosis, intraluminal debris, and inflammatory cells. Lung alveoli (a) of the WT-infected ferrets are lined with interstitial septa (arrows) and have mild to moderate inflammatory cell infiltrates. The lung alveolar architecture of the ferrets inoculated with the E119A mutant is lost due to necrosis of alveolar pneumocytes and the interstitial septa. Lung alveoli of the N294S-infected ferrets are filled with inflammatory cells and hyperplastic pneumocytes obscuring interstitial septa. Magnification, ×20.