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      Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

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          Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.

          Author Summary

          Influenza type A viruses are endemic in aquatic birds but can cross the species barrier to infect the human respiratory tract. While transmission from birds to humans is rare, the introduction of novel avian influenza viruses into immunologically naïve human populations has significant pandemic potential. Avian influenza viruses are adapted for growth at 40°C, the temperature of the avian enteric tract. However, the human proximal airways, the likely site of initial inoculation by influenza viruses, are maintained at a cooler temperature (32°C), suggesting that zoonotic transmission may be limited by temperature differences between the two hosts. Using an in vitro model of human ciliated airway epithelium, we show that avian influenza viruses grow well at 37°C, a temperature reflective of distal airways, but are restricted for infection at 32°C. A panel of genetically manipulated human influenza viruses possessing avian or avian-like surface glycoproteins were also restricted at 32°C, but not 37°C, suggesting that avian virus glycoproteins are not adapted for efficient infection at the temperature of the proximal airways. Thus, avian influenza virus infection is restricted in the human proximal airways due to the cooler temperature of this region, thus limiting the likelihood of zoonotic and subsequent human-to-human transmission of these viruses.

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          Most cited references 41

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          Avian flu: influenza virus receptors in the human airway.

          Although more than 100 people have been infected by H5N1 influenza A viruses, human-to-human transmission is rare. What are the molecular barriers limiting human-to-human transmission? Here we demonstrate an anatomical difference in the distribution in the human airway of the different binding molecules preferred by the avian and human influenza viruses. The respective molecules are sialic acid linked to galactose by an alpha-2,3 linkage (SAalpha2,3Gal) and by an alpha-2,6 linkage (SAalpha2,6Gal). Our findings may provide a rational explanation for why H5N1 viruses at present rarely infect and spread between humans although they can replicate efficiently in the lungs.
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            Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus.

            The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian alpha2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human alpha2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04 H5 HA framework, permitted binding to a natural human alpha2-6 glycan, which suggests a path for this H5N1 virus to gain a foothold in the human population.
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              A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.

              The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.

                Author and article information

                Role: Editor
                PLoS Pathog
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                May 2009
                May 2009
                15 May 2009
                : 5
                : 5
                [1 ]Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [2 ]Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                [3 ]Laboratory of Infectious Diseases, Respiratory Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, United States of America
                [4 ]Department of Virology, Division of Investigative Science, Faculty of Medicine, Imperial College London, St. Mary's Campus, London, United Kingdom
                [5 ]Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America
                Erasmus Medical Center, The Netherlands
                Author notes

                Conceived and designed the experiments: MAS LGR KS WSB RJP. Performed the experiments: MAS LGR CS KLR. Analyzed the data: MAS LGR EB RJP. Contributed reagents/materials/analysis tools: KLR WSB. Wrote the paper: MAS WSB RJP.

                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                Page count
                Pages: 13
                Research Article
                Infectious Diseases/Respiratory Infections
                Infectious Diseases/Viral Infections

                Infectious disease & Microbiology


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