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Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, Hiramatsu H, Sriwilaijaroen N, Takagi T, Suzuki Y, Ikuta K - PLoS Pathog. (2011)

Bottom Line: The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity.Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity.Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka, Japan. nabe@biken.osaka-u.ac.jp

ABSTRACT
Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

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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).
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ppat-1002068-g009: 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).

Mentions: Lungs of mice infected with 3×104 FFU viruses were examined by histopathology at 7 d post-infection. Mice infected with rEG/D1Q192H, rEG/D1129Δ,I151T or rEG/D1-EG/12 HA had much more dramatic pathological changes in their pulmonary airways and parenchymal tissues. The lungs had moderate to severe bronchiolar necrosis and alveolitis with associated hyperplasia, pulmonary edema and inflammatory cell infiltrates (Figure 9C–9E). In contrast, lung pathology of rEG/D1 and rEG/D1-EG/12 HAH192Q infected mice showed signs of limited lymphohistiocytic cell extravasations (Figure 9B and 9F). Mock-infected mice did not have lesions in their lungs (Figure 9A). H5 antigen was more extensively detected by immunohistochemistry in the alveolar area of lungs infected with rEG/D1Q192H, rEG/D1129Δ,I151T or rEG/D1-EG/12 HA than in lungs infected with rEG/D1 and rEG/D1-EG/12 HAH192Q (Figure 9G–9L). Weak antigen staining was only rarely detected in the bronchiolar area in lungs of mice infected with rEG/D1 and rEG/D1-EG/12 HAH192Q (see insert in Figure 9H and 9L). Therefore, the difference in lethality in mice infected with these viruses was grossly correlated with the growth kinetics and cytopathicity of the viruses in human airway epithelial cells. Collectively, these results indicated that enhanced receptor specificity in vivo enabled rEG/D1Q192H, rEG/D1129Δ,I151T and rEG/D1-EG/12 HA to infect mice at lower titers than rEG/D1 and rEG/D1-EG/12 HAH192Q.


Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt.

Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, Hiramatsu H, Sriwilaijaroen N, Takagi T, Suzuki Y, Ikuta K - PLoS Pathog. (2011)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102706&req=5

ppat-1002068-g009: 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).
Mentions: Lungs of mice infected with 3×104 FFU viruses were examined by histopathology at 7 d post-infection. Mice infected with rEG/D1Q192H, rEG/D1129Δ,I151T or rEG/D1-EG/12 HA had much more dramatic pathological changes in their pulmonary airways and parenchymal tissues. The lungs had moderate to severe bronchiolar necrosis and alveolitis with associated hyperplasia, pulmonary edema and inflammatory cell infiltrates (Figure 9C–9E). In contrast, lung pathology of rEG/D1 and rEG/D1-EG/12 HAH192Q infected mice showed signs of limited lymphohistiocytic cell extravasations (Figure 9B and 9F). Mock-infected mice did not have lesions in their lungs (Figure 9A). H5 antigen was more extensively detected by immunohistochemistry in the alveolar area of lungs infected with rEG/D1Q192H, rEG/D1129Δ,I151T or rEG/D1-EG/12 HA than in lungs infected with rEG/D1 and rEG/D1-EG/12 HAH192Q (Figure 9G–9L). Weak antigen staining was only rarely detected in the bronchiolar area in lungs of mice infected with rEG/D1 and rEG/D1-EG/12 HAH192Q (see insert in Figure 9H and 9L). Therefore, the difference in lethality in mice infected with these viruses was grossly correlated with the growth kinetics and cytopathicity of the viruses in human airway epithelial cells. Collectively, these results indicated that enhanced receptor specificity in vivo enabled rEG/D1Q192H, rEG/D1129Δ,I151T and rEG/D1-EG/12 HA to infect mice at lower titers than rEG/D1 and rEG/D1-EG/12 HAH192Q.

Bottom Line: The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity.Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity.Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Research Institute for Microbial Diseases (BIKEN), Osaka University, Osaka, Japan. nabe@biken.osaka-u.ac.jp

ABSTRACT
Highly pathogenic avian influenza A virus subtype H5N1 is currently widespread in Asia, Europe, and Africa, with 60% mortality in humans. In particular, since 2009 Egypt has unexpectedly had the highest number of human cases of H5N1 virus infection, with more than 50% of the cases worldwide, but the basis for this high incidence has not been elucidated. A change in receptor binding affinity of the viral hemagglutinin (HA) from α2,3- to α2,6-linked sialic acid (SA) is thought to be necessary for H5N1 virus to become pandemic. In this study, we conducted a phylogenetic analysis of H5N1 viruses isolated between 2006 and 2009 in Egypt. The phylogenetic results showed that recent human isolates clustered disproportionally into several new H5 sublineages suggesting that their HAs have changed their receptor specificity. Using reverse genetics, we found that these H5 sublineages have acquired an enhanced binding affinity for α2,6 SA in combination with residual affinity for α2,3 SA, and identified the amino acid mutations that produced this new receptor specificity. Recombinant H5N1 viruses with a single mutation at HA residue 192 or a double mutation at HA residues 129 and 151 had increased attachment to and infectivity in the human lower respiratory tract but not in the larynx. These findings correlated with enhanced virulence of the mutant viruses in mice. Interestingly, these H5 viruses, with increased affinity to α2,6 SA, emerged during viral diversification in bird populations and subsequently spread to humans. Our findings suggested that emergence of new H5 sublineages with α2,6 SA specificity caused a subsequent increase in human H5N1 influenza virus infections in Egypt, and provided data for understanding the virus's pandemic potential.

Show MeSH
Related in: MedlinePlus