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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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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.
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ppat-1002068-g007: 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.

Mentions: To examine whether the HA mutations enabling α2,6 SA binding also affected virus replication in human airway cells, we studied virus growth in primary human small airway epithelial cells (SAEC) by infecting these cells with selected recombinant viruses and human H3N2 virus, which was used as a control, at a multiplicity of infection (MOI) of 1 or 0.1 and monitoring viral growth kinetics and cytopathicity for 72 h post-infection. For comparison, we studied viral growth kinetics in chicken embryo fibroblast (CEF) cells infected at an MOI of 0.1 or 0.01. All viruses replicated well in CEF cells and produced >107 focus-forming units (FFU)/ml at 24 and 48 h post-infection. The difference in titers of these viruses was <1 log FFU/ml at each time point, indicating that all of the viruses replicated equally well in avian-derived cells (Figure 7A). These results confirmed that there was no incompatibility between EG/12 HA and EG/D1 NA or between mutated EG/D1 HA and EG/D1 NA in the recombinant viruses generated for this study (compare the kinetics of parental EG/D1 and rEG/D1 viruses in Figure 7A). In contrast, in SAEC cells (Figure 7B), rEG/D1Q192H, rEGD1129Δ,I151T and rEG/D1-EG/12 HA replicated more efficiently than rEG/D1 and rEG/D1-EG/12 HAH192Q, with slight differences in their growth, and a final virus titer of rEG/D1Q192H > rEG/D1129Δ,I151T > rEG/D1-EG/12 HA. These viruses replicated in SAEC cells and reached titers more similar to those of human H3N2 virus than of parental EG/D1, especially at a higher inoculum. The difference in virus growth kinetics correlated with cytopathicity in SAEC cells: rEG/D1Q192H, rEG/D1129Δ,I151T and rEG/D1-EG/12 HA produced more severe cytopathic effects and resulted in more detachment of infected cells at 24, 48 and 72 h post-infection than rEG/D1 and rEG/D1-EG/12 HAH192Q (Figure 7C). These results indicated that the Q192H mutant and the 129Δ/I151T double mutant produced a substantial viral growth advantage in human airway epithelial cells.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102706&req=5

ppat-1002068-g007: 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.
Mentions: To examine whether the HA mutations enabling α2,6 SA binding also affected virus replication in human airway cells, we studied virus growth in primary human small airway epithelial cells (SAEC) by infecting these cells with selected recombinant viruses and human H3N2 virus, which was used as a control, at a multiplicity of infection (MOI) of 1 or 0.1 and monitoring viral growth kinetics and cytopathicity for 72 h post-infection. For comparison, we studied viral growth kinetics in chicken embryo fibroblast (CEF) cells infected at an MOI of 0.1 or 0.01. All viruses replicated well in CEF cells and produced >107 focus-forming units (FFU)/ml at 24 and 48 h post-infection. The difference in titers of these viruses was <1 log FFU/ml at each time point, indicating that all of the viruses replicated equally well in avian-derived cells (Figure 7A). These results confirmed that there was no incompatibility between EG/12 HA and EG/D1 NA or between mutated EG/D1 HA and EG/D1 NA in the recombinant viruses generated for this study (compare the kinetics of parental EG/D1 and rEG/D1 viruses in Figure 7A). In contrast, in SAEC cells (Figure 7B), rEG/D1Q192H, rEGD1129Δ,I151T and rEG/D1-EG/12 HA replicated more efficiently than rEG/D1 and rEG/D1-EG/12 HAH192Q, with slight differences in their growth, and a final virus titer of rEG/D1Q192H > rEG/D1129Δ,I151T > rEG/D1-EG/12 HA. These viruses replicated in SAEC cells and reached titers more similar to those of human H3N2 virus than of parental EG/D1, especially at a higher inoculum. The difference in virus growth kinetics correlated with cytopathicity in SAEC cells: rEG/D1Q192H, rEG/D1129Δ,I151T and rEG/D1-EG/12 HA produced more severe cytopathic effects and resulted in more detachment of infected cells at 24, 48 and 72 h post-infection than rEG/D1 and rEG/D1-EG/12 HAH192Q (Figure 7C). These results indicated that the Q192H mutant and the 129Δ/I151T double mutant produced a substantial viral growth advantage in human airway epithelial cells.

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