David F. Burke

Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans

Generation of Swine Flu As the newly emerged influenza virus starts its journey to infect the worlds human population, the genetic secrets of the 2009 outbreak of swine influenza A(H1N1) are being revealed. In extensive phylogenetic analyses, Garten et al. (p. 197, published online 22 May) confirm that of the eight elements of the virus, the basic components encoded by the hemagglutinin, nucleoprotein, and nonstructural genes originated in birds and transferred to pigs in 1918. Subsequently, these formed a triple reassortant with the RNA polymerase PB1 that transferred from birds in 1968 to humans and then to pigs in 1998, coupled with RNA polymerases PA and PB2 that transferred from birds to pigs in 1998. The neuraminidase and matrix protein genes that complete the virus came from birds and entered pigs in 1979. The analysis offers insights into drug susceptibility and virulence, as well as raising the possibility of hitherto unknown factors determining host specificity. A significant question is, what is the potential for the H1 component of the current seasonal flu vaccine to act as a booster? Apart from the need for ongoing sequencing to monitor for the emergence of new reassortants, future pig populations need to be closely monitored for emerging influenza viruses. Evolutionary analysis suggests a triple reassortant avian-to-pig origin for the 2009 influenza A(H1N1) outbreak. Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).

Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets

Avian flu can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host. Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Crystal Structure of Fibroblast Growth Factor Receptor Ectodomain Bound to Ligand and Heparin

Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF–heparin and FGF–FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1–FGFR2–heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.

Substitutions Near the Receptor Binding Site Determine Major Antigenic Change During Influenza Virus Evolution

Flu Drift Limited Five antigenic sites in the virus surface hemagglutinin protein, which together comprise 131 amino acid positions, are thought to determine the full scope of antigenic drift of influenza A virus. Koel et al. (p. 976) show that major antigenic change can be caused by single amino acid substitutions. These single substitutions substantially skew the way the immune system “sees” the virus. All substitutions of importance are located next to the receptor-binding site in the hemagglutinin. Because there are few positions of importance for antigenic drift, there are strict biophysical limitations to the substitutions at these positions, which restricts the number of new antigenic drift variants at any point in time. Thus, the evolution of influenza virus may be more predictable than previously thought. The major antigenic changes of the influenza virus are primarily caused by a single amino acid near the receptor binding site. The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions, which occurred at only seven positions in HA immediately adjacent to the receptor binding site. Most of these substitutions were involved in antigenic change more than once. Equivalent positions were responsible for the recent antigenic changes of influenza B and A/H1N1 viruses. Substitution of a single amino acid at one of these positions substantially changed the virus-specific antibody response in infected ferrets. These findings have potentially far-reaching consequences for understanding the evolutionary mechanisms that govern influenza viruses.

The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host

Some natural influenza viruses need only three amino acid substitutions to acquire airborne transmissibility between mammals. Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

ABSTRACT The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.

Fibroblast growth factor receptors: lessons from the genes

The fibroblast growth factor receptors (FGFRs) are a family of transmembrane tyrosine kinases involved in signalling via interactions with the family of fibroblast growth factors (FGFs). Genetic findings have provided a way of dissecting these interactions. Mutations in three members of the FGFR family have been found in patients with birth defects involving craniosynostosis (premature fusion of the cranial sutures) or skeletal abnormalities. Analyses of the spectrum of mutations found predict that many of them will result in ligand-independent activation of the receptors. Amino acids have also been identified that are likely to be important in determining the specificity of FGFR-FGF interactions.

Limited airborne transmission of H7N9 influenza A virus between ferrets

Wild waterfowl form the main reservoir of influenza A viruses, from which transmission occurs directly or indirectly to various secondary hosts, including humans. Direct avian-to-human transmission has been observed for viruses of subtypes A(H5N1), A(H7N2), A(H7N3), A(H7N7), A(H9N2) and A(H10N7) upon human exposure to poultry, but a lack of sustained human-to-human transmission has prevented these viruses from causing new pandemics. Recently, avian A(H7N9) viruses were transmitted to humans, causing severe respiratory disease and deaths in China. Because transmission via respiratory droplets and aerosols (hereafter referred to as airborne transmission) is the main route for efficient transmission between humans, it is important to gain an insight into airborne transmission of the A(H7N9) virus. Here we show that although the A/Anhui/1/2013 A(H7N9) virus harbours determinants associated with human adaptation and transmissibility between mammals, its airborne transmissibility in ferrets is limited, and it is intermediate between that of typical human and avian influenza viruses. Multiple A(H7N9) virus genetic variants were transmitted. Upon ferret passage, variants with higher avian receptor binding, higher pH of fusion, and lower thermostability were selected, potentially resulting in reduced transmissibility. This A(H7N9) virus outbreak highlights the need for increased understanding of the determinants of efficient airborne transmission of avian influenza viruses between mammals.

Ab Initio Construction of Polypeptide Fragments: Accuracy of Loop Decoy Discrimination by an All-Atom Statistical Potential and the AMBER Force Field With the Generalized Born Solvation Model

The accuracy of model selection from decoy ensembles of protein loop conformations was explored by comparing the performance of the Samudrala–Moult all‐atom statistical potential (RAPDF) and the AMBER molecular mechanics force field, including the Generalized Born/surface area solvation model. Large ensembles of consistent loop conformations, represented at atomic detail with idealized geometry, were generated for a large test set of protein loops of 2 to 12 residues long by a novel ab initio method called RAPPER that relies on fine‐grained residue‐specific phi/psi propensity tables for conformational sampling. Ranking the conformers on the basis of RAPDF scores resulted in selected conformers that had an average global, non‐superimposed RMSD for all heavy mainchain atoms ranging from 1.2 Å for 4‐mers to 2.9 Å for 8‐mers to 6.2 Å for 12‐mers. After filtering on the basis of anchor geometry and RAPDF scores, ranking by energy minimization of the AMBER/GBSA potential energy function selected conformers that had global RMSD values of 0.5 Å for 4‐mers, 2.3 Å for 8‐mers, and 5.0 Å for 12‐mers. Minimized fragments had, on average, consistently lower RMSD values (by 0.1 Å) than their initial conformations. The importance of the Generalized Born solvation energy term is reflected by the observation that the average RMSD accuracy for all loop lengths was worse when this term is omitted. There are, however, still many cases where the AMBER gas‐phase minimization selected conformers of lower RMSD than the AMBER/GBSA minimization. The AMBER/GBSA energy function had better correlation with RMSD to native than the RAPDF. When the ensembles were supplemented with conformations extracted from experimental structures, a dramatic improvement in selection accuracy was observed at longer lengths (average RMSD of 1.3 Å for 8‐mers) when scoring with the AMBER/GBSA force field. This work provides the basis for a promising hybrid approach of ab initio and knowledge‐based methods for loop modeling. Proteins 2003;51:21–40.

Structural biology and bioinformatics in drug design: opportunities and challenges for target identification and lead discovery

Impressive progress in genome sequencing, protein expression and high-throughput crystallography and NMR has radically transformed the opportunities to use protein three-dimensional structures to accelerate drug discovery, but the quantity and complexity of the data have ensured a central place for informatics. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding.