The Wilhelmine E. Key 1999 Invitational lecture. Predicting the evolution of human influenza A.

We studied the evolution of the HA1 domain of the H3 hemagglutinin gene from human influenza virus type A. The phylogeny of these genes showed a single dominant lineage persisting over time. We tested the hypothesis that the progenitors of this single evolutionarily successful lineage were viruses carrying mutations at codons at which prior mutations had helped the virus to avoid human immune surveillance. We found evidence that eighteen hemagglutinin codons appeared to have been under positive selection to change the amino acid they encoded in the past. Retrospective tests show that viral lineages undergoing the greatest number of mutations in the positively selected codons were the progenitors of future H3 lineages in nine of eleven recent influenza seasons. Codons under positive selection were associated with antibody combining sites A or B or the sialic acid receptor binding site. However, not all codons in these sites had predictive value. Monitoring new H3 isolates for additional changes in positively selected codons might help identify the most fit extant viral strains that arise during antigenic drift.

Predicting the evolution of human influenza A.

Eighteen codons in the HA1 domain of the hemagglutinin genes of human influenza A subtype H3 appear to be under positive selection to change the amino acid they encode. Retrospective tests show that viral lineages undergoing the greatest number of mutations in the positively selected codons were the progenitors of future H3 lineages in 9 of 11 recent influenza seasons. Codons under positive selection were associated with antibody combining site A or B or the sialic acid receptor binding site. However, not all codons in these sites had predictive value. Monitoring new H3 isolates for additional changes in positively selected codons might help identify the most fit extant viral strains that arise during antigenic drift.

Positive selection on the H3 hemagglutinin gene of human influenza virus A.

The hemagglutinin (HA) gene of influenza viruses encodes the major surface antigen against which neutralizing antibodies are produced during infection or vaccination. We examined temporal variation in the HA1 domain of HA genes of human influenza A (H3N2) viruses in order to identify positively selected codons. Positive selection is defined for our purposes as a significant excess of nonsilent over silent nucleotide substitutions. If past mutations at positively selected codons conferred a selective advantage on the virus, then additional changes at these positions may predict which emerging strains will predominate and cause epidemics. We previously reported that a 38% excess of mutations occurred on the tip or terminal branches of the phylogenetic tree of 254 HA genes of influenza A (H3N2) viruses. Possible explanations for this excess include processes other than viral evolution during replication in human hosts. Of particular concern are mutations that occur during adaptation of viruses for growth in embryonated chicken eggs in the laboratory. Because the present study includes 357 HA sequences (a 40% increase), we were able to separately analyze those mutations assigned to internal branches. This allowed us to determine whether mutations on terminal and internal branches exhibit different patterns of selection at the level of individual codons. Additional improvements over our previous analysis include correction for a skew in the distribution of amino acid replacements across codons and analysis of a population of phylogenetic trees rather than a single tree. The latter improvement allowed us to ascertain whether minor variation in tree structure had a significant effect on our estimate of the codons under positive selection. This method also estimates that 75.6% of the nonsilent mutations are deleterious and have been removed by selection prior to sampling. Using the larger data set and the modified methods, we confirmed a large (40%) excess of changes on the terminal branches. We also found an excess of changes on branches leading to egg-grown isolates. Furthermore, 9 of the 18 amino acid codons, identified as being under positive selection to change when we used only mutations assigned to internal branches, were not under positive selection on the terminal branches. Thus, although there is overlap between the selected codons on terminal and internal branches, the codons under positive selection on the terminal branches differ from those on the internal branches. We also observed that there is an excess of positively selected codons associated with the receptor-binding site and with the antibody-combining sites. This association may explain why the positively selected codons are restricted in their distribution along the sequence. Our results suggest that future studies of positive selection should focus on changes assigned to the internal branches, as certain of these changes may have predictive value for identifying future successful epidemic variants.

Differences in the biological phenotype of low-yielding (L) and high-yielding (H) variants of swine influenza virus A/NJ/11/76 are associated with their different receptor-binding activity.

Low- (L) and high-yielding (H) variants of A/sw/NJ/11/76 influenza virus were compared for their growth properties in embryonated chicken eggs and MDCK cells and for their binding affinity for the membrane fractions prepared from cells of the chicken embryo allantoic membrane. MDCK, and swine tracheal cells, as well as for soluble sialic acid containing macromolecules and monovalent sialosides. We have shown, that during infection in MDCK cells and in eggs, the progeny of the L variant remain predominantly cell associated, in contrast to those of H. As a result, accumulation of the L mutant in allantoic or culture fluid is significantly slowed in comparison with the H variant. Visualization of the infectious foci formed by the viruses in MDCK cell monolayers and on the allantoic membrane revealed that L spreads predominantly from cell to cell, while the spread of H involves release of the virus progeny into solution and its rapid distribution over the cell monolayer via convectional flow of the liquid. In the binding assays, L displayed significantly higher binding affinity than H for cellular membranes, gangliosides, and sialylglycoproteins, however, the affinity of the variants for the monovalent sialic acid compounds was comparable. Unlike H. L bound strongly to dextran sulfate. The data obtained suggest that all distinctions of the L and H biological phenotypes reported previously [Kilbourne, E.D., Taylor, A. H. Whitaker, C.W., Sahai, R., and Caton, A (1988) Hemagglutinin polymorphism as the basis for low-and high-yield phenotypes of swine influenza virus. Proc. Natl. Acad. Sci. USA 85, 7782-7785] could be rationally explained by a more avid binding of the L variant to the surface of target cells, and that this effect is mainly due to enhanced electrostatic interactions.

Long term trends in the evolution of H(3) HA1 human influenza type A.

We have studied the HA1 domain of 254 human influenza A(H3N2) virus genes for clues that might help identify characteristics of hemagglutinins (HAs) of circulating strains that are predictive of that strain's epidemic potential. Our preliminary findings include the following. (i) The most parsimonious tree found requires 1,260 substitutions of which 712 are silent and 548 are replacement substitutions. (ii) The HA1 portion of the HA gene is evolving at a rate of 5.7 nucleotide substitutions/year or 5.7 x 10(-3) substitutions/site per year. (iii) The replacement substitutions are distributed randomly across the three positions of the codon when allowance is made for the number of ways each codon can change the encoded amino acid. (iv) The replacement substitutions are not distributed randomly over the branches of the tree, there being 2.2 times more changes per tip branch than for non-tip branches. This result is independent of how the virus was amplified (egg grown or kidney cell grown) prior to sequencing or if sequencing was carried out directly on the original clinical specimen by PCR. (v) These excess changes on the tip branches are probably the result of a bias in the choice of strains to sequence and the detection of deleterious mutations that had not yet been removed by negative selection. (vi) There are six hypervariable codons accumulating replacement substitutions at an average rate that is 7.2 times that of the other varied codons. (vii) The number of variable codons in the trunk branches (the winners of the competitive race against the immune system) is 47 +/- 5, significantly fewer than in the twigs (90 +/- 7), which in turn is significantly fewer variable codons than in tip branches (175 +/- 8). (viii) A minimum of one of every 12 branches has nodes at opposite ends representing viruses that reside on different continents. This is, however, no more than would be expected if one were to randomly reassign the continent of origin of the isolates. (ix) Of 99 codons with at least four mutations, 31 have ratios of non-silent to silent changes with probabilities less than 0.05 of occurring by chance, and 14 of those have probabilities <0.005. These observations strongly support positive Darwinian selection. We suggest that the small number of variable positions along the successful trunk lineage, together with knowledge of the codons that have shown positive selection, may provide clues that permit an improved prediction of which strains will cause epidemics and therefore should be used for vaccine production.

Genetic variation in neuraminidase genes of influenza A (H3N2) viruses.

Nucleotide sequences of the neuraminidase (NA) genes of 33 influenza A (H3N2) epidemic strains isolated between 1968 and 1995 were analyzed to determine their evolutionary relationships. Phylogenetic analysis using the DNA maximum-likelihood method indicates that the NA genes of recent H3N2 field strains, like their hemagglutinin genes (HA), have evolved as two distinct lineages represented by the vaccine strains. A/Beijing/353/89 and A/Beijing/32/92 for A/Shanghai/24/ 90). Furthermore, genetic reassortment of NA genes between the two lineages occurred during their circulation. Genetic reassortants, which bear an A/Beijing/32/92-like HA and an A/Beijing/353/89-like NA, have circulated worldwide and are representative of current influenza A (H3N2) epidemic strains. The mutation rate of the NA gene was found to be 2.28 x 10(-3) per nucleotide site per year with 4.2% of the mutations resulting in amino acid substitutions. Thirty-five percent of the amino acid substitutions was located in sites previously suggested to be reactive to antibody. Amino acid residues involved in NA enzyme activity have been conserved. Seven potential glycosylation sites identified in the NA of A/Hong Kong/8/68 virus were conserved by the majority of isolates, with more recently circulating viruses having an additional glycosylation site. Comparison of the rate of amino acid substitutions in the NA stalk to that of entire NA revealed high variability in this region. These findings demonstrate the importance of closely monitoring both the HA and the NA genes of influenza viruses to aid vaccine strain selection.

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism.

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)