Figure 1.
Phylogenetic tree of HA genes of H5N1 viruses isolated in Egypt.
This tree includes published HA sequences of 85 H5N1 influenza A viruses isolated in Egypt, from the National Center for Biotechnology Information database (minimum sequence length 1,644 nt), and 21 HA sequences determined in this study (sequence length 1,707 nt). The newly analyzed sequences in this study are marked with a black circle. The strains whose HA sequences were determined in this study and were analyzed further for receptor binding specificity are marked with a red circle. The strains whose HA sequences were previously reported and were analyzed for receptor binding specificity in this study are marked with a blue circle. Colors are used to highlight virus strains with different hosts, isolation year and sublineage.
Figure 2.
Receptor-binding specificity of H5N1 viruses isolated in Egypt.
Direct binding of viruses to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acids was measured. (A) Seasonal human influenza H3N2 virus. (B) Avian influenza H5N3 virus. (C)–(H) Isolates from 2007–2009 outbreaks in Egypt. (I)–(P) Recombinant EG/D1 viruses with different HAs as indicated. Each data point is the mean ± SD of triplicate experiments.
Figure 3.
Effect of HA mutations in sublineage A viruses on receptor specificity of EG/D1 virus HA.
(A) The two mutations found in the HAs of sublineage A viruses were introduced into the HA of EG/D1 virus as single and double mutations. (B) The reverse mutations were introduced into the HA of EG/12 virus. Direct binding to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acid was assayed. Mutations are indicated by subscripts. Each data point is the mean ± SD of triplicate experiments.
Table 1.
Mutations in HA genes in H5 viruses in sublineages A, BI and BII.
Figure 4.
Effect of HA mutations in sublineage BI viruses on receptor specificity of EG/D1 HA.
(A) The mutations found in sublineage BI viral HAs were introduced as single and multiple mutations into the HA of EG/D1 virus. (B) The reverse mutations were introduced into the HA of EG/0929 virus. Direct binding to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acid was measured. Mutations are indicated by subscripts. Each data point is the mean ± SD of triplicate experiments.
Figure 5.
Effect of HA mutations in sublineage BII viruses on receptor specificity of EG/D1 HA.
The mutations found in sublineage BΙΙ viral HAs were introduced as single and multiple mutations into the HA of EG/D1 virus. Direct binding to sialylglycopolymers containing either α2,3-linked (blue) or α2,6-linked (red) sialic acid was measured. Mutations are indicated by subscripts. Each data point is the mean ± SD of triplicate experiments.
Table 2.
Prevalence of HA mutations characteristic of H5 sublineages A, BI and BII viruses in virus isolates from Egypt and Asia.
Figure 6.
Attachment of rEG/D1 viruses to tissues of the human respiratory tract.
The attachment patterns of A/Japan/434/2003 (H3N2), A/Duck/Hong Kong/820/80 (H5N3), and eight rEG/D1 viruses (rEG/D1, rEG/D1-EG/11 HA, rEG/D1-EG/29 HA, rEG/D1-EG/12 HA, rEG/D1Q192H, rEG/D1129Δ,I151T, rEG/D1129Δ,I151T,V210I and rEG/D1-EG/12 HAH192Q) to fixed human larynx, trachea and alveoli tissue sections were examined by histochemistry. Attached viruses were stained red. Arrows and arrow-heads indicate type I and type ΙΙ pneumocytes, respectively. The panels were chosen to reflect the attachment pattern in each tissue section as much as possible.
Figure 7.
Growth kinetics of rEG/D1 viruses in avian cells and human cells.
(A) CEF cells were infected in triplicate with parental EG/D1 and five rEG/D1 viruses (rEG/D1, rEG/D1Q192H, rEG/D1129Δ,I151T, rEG/D1-EG/12 HA and rEG/D1-EG/12 HAH192Q) at an MOI of 0.1 or 0.01. (B) Human SAEC cells were infected in triplicate with the viruses at an MOI of 1 or 0.1. The culture supernatants were harvested at the indicated times and assayed for focus-forming units on CEF cells to determine the progeny virus titer (log10 FFU/ml). Each data point in (A) and (B) is the mean ± SD of triplicate experiments. (C) Phase contrast microscopy of morphological changes in SAEC cells infected by the indicated viruses at an MOI of 0.1 and examined at the indicated times post-infection.
Figure 8.
Mortality and weight loss of mice infected with rEG/D1 viruses.
Six-week-old BALB/c mice (7–8 mice per group) were inoculated intranasally with the indicated doses of rEG/D1, rEG/D1Q192H, rEG/D1129Δ,I151T, rEG/D1-EG/12 HA and rEG/D1-EG/12 HAH192Q. (A) Body weight of infected mice was monitored up to 14 d post-infection. Mean percent body weight change (±SD) for each group of mice is shown. (B) Survival of mice inoculated with rEG/D1 viruses. Mortality was calculated including mice that were sacrificed because they had lost more than 30% of their body weight. (C) Virus titers in lungs of mice infected with 3×104 or 3×105 FFU rEG/D1 at the indicated times post-infection. Circles and diamonds indicate values in individual mice.
Figure 9.
Histopathology and immunohistochemistry in lung tissues of mice infected with rEG/D1 viruses.
Photomicrographs of hematoxylin-and-eosin (H&E) stained and immunohistochemically (IHC) stained lung sections from mice infected with 3×104 FFU rEG/D1 viruses 7 d post-infection are shown as follows. (A) and (G) mock-infected. (B) and (H) rEG/D1-infected. (C) and (I) rEG/D1Q192H-infected. (D) and (J) rEG/D1129Δ,I151T-infected. (E) and (K) rEG/D1-EG/12 HA-infected. (F) and (L) rEG/D1-EG/12 HAH192Q-infected. In the IHC-stained tissues, viral antigen is stained deep brown on a hematoxylin-stained background (arrows). In mice infected with rEG/D1 and rEG/D1-EG/12 HAH192Q, positive staining was detected sporadically in the bronchiolar epithelium (insert).
Figure 10.
Analysis of receptor docking modes of EG/D1 HA and HA mutants.
Structural models of H5 HA. (A) Ribbon model of EG/D1 HA. The trimeric globular-head region is shown. Key residues in our analysis are shown in a colored space-filling model. Receptor binding domains are colored blue (130 loop), green (190 helix) and purple (220 loop). (B) Molecular surface of EG/D1 HA. The red circle indicates the receptor binding pocket. (C) Docking models for EG/D1, EG/D1Q192H and EG/D1129Δ,I151T HA with a human-type receptor analog (PDBID code 1MQN). Residues 127E, 128A, 130S and 131G are colored green, as is 129S, and the other residues and domains are displayed in the same colors as above. An additional hydrogen bond between E127 and T151 is indicated in the red circle. The Udock scores of the corresponding complexes are shown at the bottom.