Acquisition of Human-Type Receptor Binding Specificity by New H5N1 Influenza Virus Sublineages during Their Emergence in Birds in Egypt

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.

Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons (Columba livia) in Egypt

“…Previously, it was proposed that isolates having isoleucine at residue 151 of the HA1 protein might be involved in different receptor binding (Claas et al, 1998). Recently, it was shown that deletion of RBD 129S combined with I151T increased α2-6 SA binding (Watanabe et al, 2011). It was observed that the pigeon strain isolated in this study has these substitutions.…”

Genesis and Dissemination of Highly Pathogenic H5N6 Avian Influenza Viruses

“…A list of amino acid substitutions of biological relevance for mammalian adaptations or reducing drug susceptibility was investigated for the three reassortant isolates (Table 1). Although the most well-known receptor binding sites (222 to 224, H5 numbering) were still avian-like (QSG) in the three reassortant isolates (21), other mutations that may alter receptor specificity were detected, such as S123P, I151T, and T156A (22)(23)(24). The reassortant C isolates carried a deletion at position 126 in the HA, adding a potential N-glycosylation at site 124.…”

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH

“…Viruses and Cells-Influenza virus strains A/crow/Kyoto/ 53/04 (H5N1) (Cw/Ky (H5N1)) and A/chicken/Egypt/CL6/07 (H5N1) (Ck/Eg (H5N1)) were isolated from embryonated chicken eggs inoculated with tracheal or lung homogenates from dead crows (10) or dead chickens (11), respectively. A/Beijing/262/95 (H1N1) (Beijing (H1N1)), A/Duck/Hong Kong/ 273/78 (H2N2) (Dk/Hk (H2N2)), A/Panama/2007/99 (H3N2) (Panama (H3N2)), A/Duck/Hong Kong/668/79 (H4N5) (Dk/ Hk (H4N5)), A/Duck/Hong Kong/820/80 (H5N3) (Dk/Hk (H5N3)), A/Turkey/Ontario/7732/66 (H5N9) (Tk/Ont (H5N9)), A/Duck/Hong Kong/960/80 (H6N2) (Dk/Hk (H6N2)), A/wigeon/ Osaka/1/01 (H7N7) (Wg/Os (H7N7)), A/Turkey/Ontario/6118/67 (H8N4) (Tk/Ont (H8N4)), and A/Duck/Hong Kong/448/78 (H9N2) (Dk/Hk (H9N2)) were kind gifts from Yoshinobu Okuno (Kanonji Institute, the Research Foundation for Microbial Diseases of Osaka University, Kagawa, Japan).…”

“…The HA sequences of A/Thailand/ Kan353/04 (H5N1) (Thailand (H5N1)), A/Indonesia/5/05 (H5N1) (Indonesia (H5N1)), and A/Shanghai/1/06 (H5N1) (Shanghai (H5N1)) were constructed by PCR using overlapping deoxyoligonucleotides corresponding to the published sequence of the HA open reading frame. The full-length sequences of both A/duck/Egypt/D1Br/2007 (H5N1) (Dk/Eg (H5N1)) (clade 2.2.1) (PB2, PB1, PA, HA, NP, NA, M, and NS) (11) and Dk/Hk (H5N3) (PB2, PB1, PA, HA, NP, NA, M, and NS) were constructed by PCR. The virulent HA sequence of Dk/Hk (H5N3) was constructed by changing single basic amino acids within the HA cleavage site to multiple basic amino acids (NЈ-TR-CЈ to NЈ-RRKKR-CЈ), as described previously (10).…”