Receptor Binding Profiles of Avian Influenza Virus Hemagglutinin Subtypes on Human Cells as a Predictor of Pandemic Potential

To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.

Section: Discussionsupporting

confidence: 57%

Analysis of Influenza Virus Hemagglutinin Receptor Binding Mutants with Limited Receptor Recognition Properties and Conditional Replication Characteristics

Bradley

Galloway

Lasanajak

et al. 2011

“…Conversely, ␣2,3-linked SA receptors are found at higher levels on nonciliated bronchiolar cells and alveolar type II cells in the lower respiratory tract (2,5,6,8). Consistent with these findings, studies of virus attachment have shown that human influenza viruses bound more abundantly to the upper respiratory tract than avian influenza viruses (2,9,10). Human influenza viruses attach primarily to ciliated epithelial cells and to a lesser extent to goblet cells in the upper respiratory tract, as well as to type I pneumocytes in the alveoli (6,10,11).…”

supporting

confidence: 73%

“…The finding that human influenza viruses mainly infected ciliated cells in our primary FTE cell model, on which ␣2,6-linked SA receptors predominate, is consistent with the tissue tropism of seasonal influenza viruses, which infect mainly the upper respiratory tract of humans and ferrets (2,9,10). FTE cultures may not be able to recapture the apparent higher density of ciliated cells present in the native ferret trachea (33), which may result in differences in sialic acid distribution.…”

Section: Discussionmentioning

confidence: 50%

“…Subsequently, it has been shown that goblet cells also express ␣2,6-linked SA receptors (8), and the human-adapted 1918 HA demonstrated substantial binding to goblet cell regions of human tracheal tissue (21,43). Conversely, studies using human tracheal epithelial cell cultures have shown that human influenza viruses infected both nonciliated cells and ciliated cells (25,29) and that avian influenza viruses were predominantly localized on ciliated cells (9,25,26,29,30). The reasons for the discrepancies in cellular tropism of influenza virus between our in vitro model of ferret tracheal differentiated epithelial cells and those found in human tracheal epithelial cells are not clear.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Tropism and Infectivity of Influenza Virus, Including Highly Pathogenic Avian H5N1 Virus, in Ferret Tracheal Differentiated Primary Epithelial Cell Cultures

Zeng

Goldsmith

Maines

et al. 2013

Tropism and adaptation of influenza viruses to new hosts is partly dependent on the distribution of the sialic acid (SA) receptors to which the viral hemagglutinin (HA) binds. Ferrets have been established as a valuable in vivo model of influenza virus pathogenesis and transmission because of similarities to humans in the distribution of HA receptors and in clinical signs of infection. In this study, we developed a ferret tracheal differentiated primary epithelial cell culture model that consisted of a layered epithelium structure with ciliated and nonciliated cells on its apical surface. We found that human-like (␣2,6-linked) receptors predominated on ciliated cells, whereas avian-like (␣2,3-linked) receptors, which were less abundant, were presented on nonciliated cells. When we compared the tropism and infectivity of three human (H1 and H3) and two avian (H1 and H5) influenza viruses, we observed that the human influenza viruses primarily infected ciliated cells and replicated efficiently, whereas a highly pathogenic avian H5N1 virus (A/Vietnam/1203/2004) replicated efficiently within nonciliated cells despite a low initial infection rate. Furthermore, compared to other influenza viruses tested, VN/1203 virus replicated more efficiently in cells isolated from the lower trachea and at a higher temperature (37°C) compared to a lower temperature (33°C). VN/1203 virus infection also induced higher levels of immune mediator genes and cell death, and virus was recovered from the basolateral side of the cell monolayer. This ferret tracheal differentiated primary epithelial cell culture system provides a valuable in vitro model for studying cellular tropism, infectivity, and the pathogenesis of influenza viruses.

“…The EIV and CIV HA ectodomains (the same sequences as in the reverse-genetics plasmids) were fused to human IgG1 Fc at the C terminus, followed by a hexahistidine tag (39,40). The baculovirus gp64 secretion peptide was fused to the constructs at the N terminus.…”

Section: Eivmentioning

confidence: 99%

Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence

Feng

Gonzalez

Deng

et al. 2015

The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred ␣2-3-over ␣2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to ␣2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. IMPORTANCEInfluenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the crossspecies transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology. Influenza A viruses are maintained in aquatic birds as intestinal infections, occasionally transfer to and become established as respiratory infections in mammals, including humans, and sometimes spread from one mammal to another (1, 2). Mammalian hosts that have been commonly seen to maintain avian-derived viruses include swine, horses, humans, mink, seals, and, recently, dogs (2-5). Host transfers between different birds, from birds to mammals, or between different mammalian hosts are relatively common but mostly result in single infe...