Abstract
H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern’ G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.Emerging Microbes & Infections (2017) 6, e11; doi:10.1038/emi.2016.139; published online 22 March 2017
Highlights
IntroductionOver the past 20 years, H9N2 avian influenza viruses (AIVs) have become enzootic in poultry throughout Asia, the Middle East and North Africa, where they have caused major economic losses to the poultry industry, as well as sporadic zoonotic human infections.[1,2,3,4] Studies to assess the mammalian transmissibility of H9N2 viruses conducted in ferrets have clearly demonstrated both direct contact and airborne transmission to naive ferrets, both when the virus was unadapted or ‘mammalian-adapted’ by serial ferret passage.[5,6,7,8] Given the transmission of H9N2 viruses in the ferret model and their widespread distribution, H9N2 AIVs are considered to be potentially pandemic viruses.Many properties of influenza virus proteins have been determined to have a role in the adaptation of avian viruses to humans
In addition to receptor binding, several studies have shown that efficient airborne transmission of avian influenza viruses (AIVs) between ferrets requires the HA molecule to become stabilized and exhibit a lower pH of fusion.[9,10,19,20,21]. This increased pH stability of the HA is thought to be important for maintaining viral infectivity in the relatively harsh microenvironment of respiratory droplets and the mildly acidic environment of the mammalian nasal tract and human influenza viruses generally have a lower pH of fusion, whereas AIVs have a higher pH of fusion (4pH 5.5).[19,22]
We measured binding to the avian and human receptor analogues, 3′-sialylacetyllactosamine (3SLN) and 6′-sialylacetyllactosamine (6SLN), as well as to a sulphated version of the avian analogue 3SLN, Neu5Ac α2,3Gal β1-4(6-HSO3)GlcNAc (hereafter referred to as 3SLN(6su)) that has been implicated in AIV receptor binding.[35,36]
Summary
Over the past 20 years, H9N2 avian influenza viruses (AIVs) have become enzootic in poultry throughout Asia, the Middle East and North Africa, where they have caused major economic losses to the poultry industry, as well as sporadic zoonotic human infections.[1,2,3,4] Studies to assess the mammalian transmissibility of H9N2 viruses conducted in ferrets have clearly demonstrated both direct contact and airborne transmission to naive ferrets, both when the virus was unadapted or ‘mammalian-adapted’ by serial ferret passage.[5,6,7,8] Given the transmission of H9N2 viruses in the ferret model and their widespread distribution, H9N2 AIVs are considered to be potentially pandemic viruses.Many properties of influenza virus proteins have been determined to have a role in the adaptation of avian viruses to humans. Over the past 20 years, H9N2 avian influenza viruses (AIVs) have become enzootic in poultry throughout Asia, the Middle East and North Africa, where they have caused major economic losses to the poultry industry, as well as sporadic zoonotic human infections.[1,2,3,4] Studies to assess the mammalian transmissibility of H9N2 viruses conducted in ferrets have clearly demonstrated both direct contact and airborne transmission to naive ferrets, both when the virus was unadapted or ‘mammalian-adapted’ by serial ferret passage.[5,6,7,8] Given the transmission of H9N2 viruses in the ferret model and their widespread distribution, H9N2 AIVs are considered to be potentially pandemic viruses. The molecular basis of this change in receptor preference has been previously evaluated for several influenza subtypes (e.g., H2, H3, H5, H7 and H9) and has been partially, or entirely, attributed to a single amino-acid change at residue 226 in the HA molecule from glutamine to leucine.[13,14,15,16] In an H9N2 isolate, a Q226L mutation alone allowed the virus to replicate better in human primary epithelial airway cells and L226 has been shown to be structurally involved in the interaction with a α2,6 receptor analogue.[17,18] In addition to receptor binding, several studies have shown that efficient airborne transmission of AIV between ferrets requires the HA molecule to become stabilized and exhibit a lower pH of fusion.[9,10,19,20,21] This increased pH stability of the HA is thought to be important for maintaining viral infectivity in the relatively harsh microenvironment of respiratory droplets and the mildly acidic environment of the mammalian nasal tract and human influenza viruses generally have a lower pH of fusion (opH 5.5), whereas AIVs have a higher pH of fusion (4pH 5.5).[19,22]
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