Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody

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: Discussionmentioning

confidence: 99%

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

Bradley

Galloway

Lasanajak

et al. 2011

“…Furthermore, site 234 (226 in H3 numbering) falls within epitope II of H9 and plays a role in the receptor binding specificity of HA (20). (20,35) and glycosylation in these two sites could play a role in antigenic variation. Similarly, in the NA gene, we identified seven residues under positive selection pressure, six of which were located in the NA globular head (25).…”

Section: Phylogeography Of H9n2 In Central Asia and The Middlementioning

confidence: 99%

Phylogeography and Evolutionary History of Reassortant H9N2 Viruses with Potential Human Health Implications

Fusaro

Monne

Salviato

et al. 2011

149

153

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far andMiddle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter-and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.

“…Virus rescue was performed as previously described (24,39). Briefly, DNA and Lipofectamine 2000 (Invitrogen) were mixed (2 l of transfection reagent per 1 g of DNA), incubated at room temperature for 30 min, and added to an 80 to 90% confluent monolayer of 293T cells in six-well plates.…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

PB2 and Hemagglutinin Mutations Are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus

Ping

Dankar

Forbes

et al. 2010

Self Cite

Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >10 3.8 -fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD 50 ] >101.4 -fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (10 2 -fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (10 0.3 -to 10 0.8 -fold), and undetectable effects on the LD 50 for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA.