Katsuhisa Nakajima

Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses☆

We determined the sequences of 7 serotypes (H4, H6, H8, H9, H11, H12, and H13) of hemagglutinin (HA) genes, which have not been reported so far. The coding regions consisted of 1692 nucleotides in H4, 1698 in H6, 1695 in H8, 1680 in H9, 1695 in H11, 1692 in H12, and 1698 in H13, and specified 564, 566, 565, 560, 565, 564, and 566 amino acids, respectively. By comparison of amino acid sequences, 13 HA serotypes could be divided into two families, i.e., an H1 group (H1, H2, H5, H6, H8, H9, H11, H12, and H13) and an H3 group (H3, H4, H7, and H10). The relationship was essentially similar to that reported by Air from the comparison of 80 amino-terminal amino acid sequence of 12 HA serotypes (G.M. Air, 1981, Proc. Natl. Acad. Sci. USA 78, 7639-7643). Though a considerable amino acid sequence difference exists between certain HA serotypes, several amino acid residues in fusion peptides (HA2(1-11)) and receptor-binding sites (HA1(98), -134, -138, -153, -183, and -195) were shown to be conserved among the 13 HA serotypes. Human H1 and avian H3, H4, H8, and H10 viruses preferentially bound NeuAc alpha 2,3Gal sequences, whereas human H2 and H3 and avian H6 and H9 viruses bound NeuAc alpha 2,6Gal sequences, although the amino acid residues at position 226 of human H2 and avian H6 and H9 serotype HAs are glutamine. These results show that the amino acid residue at position 226 is not necessarily a determinant of receptor specificity for all serotypes.

Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

Outbreaks of Norwalk-like virus-associated gastroenteritis traced to shellfish: coexistence of two genotypes in one specimen.

We determined the nucleotide sequences of Norwalk-like viruses in 10 PCR products from stool or oyster specimens obtained from four outbreaks of gastroenteritis in which shellfish was suspected as the cause in Shizuoka prefecture in Japan between 1987-94. The sequences were determined from nucleotide positions 4561-4852 (292 bp) in the polymerase region. Two types of sequences were detected. One (genotype 1) had 87% sequence homology with the prototype Norwalk virus, and the other (genotype 2) had 59% sequence homology. The sequences from isolates belonging to the same genotype were almost the same regardless of the year of isolation. Because sequences of 2 genotypes were detected in 2 of the 4 outbreaks, nested PCR was performed with genotype-specific primers to detect the presence of 2 genotypes in the same specimen. In 5 of 10 specimens, PCR bands were detected with both genotype-specific primers, indicating the coexistence of 2 genotypes in 1 specimen. We also detected two genotypes of Norwalk-like virus in an oyster from a sample implicated in one of the outbreaks which may provide direct evidence of oysters as the cause of the gastroenteritis.

Inhibitory effect of protein kinase C inhibitor on the replication of influenza type A virus

The growth of influenza virus A/PR/8/34 in MDCK cells was inhibited by 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H7) which is a potent inhibitor of protein kinase C, but not by an effective inhibitor of cyclic nucleotide-dependent protein kinases. Analysing the inhibitory effect of H7 during the replication cycle of influenza virus, we found that the primary transcripts were sufficiently synthesized in infected cells exposed to H7. The primary transcripts synthesized in the presence and absence of H7 were active in directing the synthesis of viral polypeptides both in a cell-free system and in the system containing H7. In the system where infected cells were exposed to H7, the viral positive-sense RNAs were also significantly amplified 6 h after infection. However, the synthesis of viral proteins other than nucleoprotein from viral primary or amplified (secondary) mRNAs was extremely restricted. The synthesis of host cellular proteins in mock-infected cells was significantly retained in the presence of H7. These results suggest that the selective inhibition of influenza virus translation following the transcription of viral mRNA was induced by H7 in infected cells.

Detection of influenza viruses in throat swab by using polymerase chain reaction

An assay protocal based on exploiting the polymerase chain reaction (PCR) for the direct detection of influenza virus in throat swab is described. By use of the mixture of HI and H3 primers, it was possible to determine the subtype of the influenza A viruses simultaneously. No visible band was detected after PCR of influenza B or A (H2N2) viruses with a pair of HI or H3 primers. The dilution experiment showed that the influenza viruses, as few as 1.3–6 plaque‐forming units, were sufficient for detecting the HA gene by PCR. All throat swab samples from which influenza viruses had been isolated by conventional method were also positively detected by PCR method.

Theoretical analysis of binding specificity of influenza viral hemagglutinin to avian and human receptors based on the fragment molecular orbital method

The hemagglutinin (HA) protein of the influenza virus binds to the host cell receptor in the early stage of viral infection. A change in binding specificity from avian 2-3 to human 2-6 receptor is essential for optimal human-to-human transmission and pandemics. Therefore, it is important to reveal the key factors governing the binding affinity of HA-receptor complex at the molecular level for the understanding and prediction of influenza pandemics. In this work, on the basis of ab initio fragment molecular orbital (FMO) method, we have carried out the interaction energy analysis of HA-receptor complexes to quantitatively elucidate the binding specificity of HAs to avian and human receptors. To discuss the binding property of influenza HA comprehensively, a number of HAs from human H1, swine H1, avian H3 and avian H5 viruses were analyzed. We performed detailed investigations about the interaction patterns of complexes of various HAs and receptor analogues, and revealed that intra-molecular interactions between conserved residues in HA play an important role for HA-receptor binding. These results may provide a hint to understand the role of conserved acidic residues at the receptor binding site which are destabilized by the electrostatic repulsion with sialic acid. The calculated binding energies and interaction patterns between receptor and HAs are consistent with the binding specificities of each HA and thus explain the receptor binding mechanism. The calculated results in the present analysis have provided a number of viewpoints regarding the models for the HA-receptor binding specificity associated with mutated residues. Examples include the role of Glu190 and Gln226 for the binding specificity of H5 HA. Since H5 HA has not yet been adapted to human receptor and the mechanism of the specificity change is unknown, this result is helpful for the prediction of the change in receptor specificity associated with forthcoming possible pandemics.

Possibility of Mutation Prediction of Influenza Hemagglutinin by Combination of Hemadsorption Experiment and Quantum Chemical Calculation for Antibody Binding

We have performed a quantum-chemical MP2/6-31G* calculation for the hemagglutinin (HA) antigen-antibody system of the H3N2 influenza virus with the fragment molecular orbital method, which provides one of the worlds largest ab initio electron-correlated calculations for biomolecular systems. On the basis of the calculated interfragment interaction energies (IFIEs) representing the molecular interactions between the amino acid residues in the antigen-antibody complex, we have identified those residues in the antigenic region E of HA protein that are significantly recognized by the Fab fragment of antibody with strongly attractive interactions. Combining these IFIE results with those of hemadsorption experiments by which the mutation-prohibited sites are specified has enabled us to explain most of the historical mutation data (five of six residues), which would thus provide a promising method for predicting the HA residues that have a high probability of forthcoming mutation.

Accumulation of Amino Acid Substitutions Promotes Irreversible Structural Changes in the Hemagglutinin of Human Influenza AH3 Virus during Evolution

ABSTRACT In order to clarify the effect of an accumulation of amino acid substitutions on the hemadsorption character of the influenza AH3 virus hemagglutinin (HA) protein, we introduced single-point amino acid changes into the HA1 domain of the HA proteins of influenza viruses isolated in 1968 (A/Aichi/2/68) and 1997 (A/Sydney/5/97) by using PCR-based random mutation or site-directed mutagenesis. These substitutions were classified as positive or negative according to their effects on the hemadsorption activity. The rate of positive substitutions was about 50% for both strains. Of 44 amino acid changes that were identical in the two strains with regard to both the substituted amino acids and their positions in the HA1 domain, 22% of the changes that were positive in A/Aichi/2/68 were negative in A/Sydney/5/97 and 27% of the changes that were negative in A/Aichi/2/68 were positive in A/Sydney/5/97. A similar discordance rate was also seen for the antigenic sites. These results suggest that the accumulation of amino acid substitutions in the HA protein during evolution promoted irreversible structural changes and therefore that antigenic changes in the H3HA protein may not be limited.

Studies on the molecular basis for loss of the ability of recent influenza A (H1N1) virus strains to agglutinate chicken erythrocytes.

Recent strains of influenza A but not B viruses have lost the ability to agglutinate chicken red blood cells (CRBC). The H1N1 viruses isolated in Japan during the 1991/92 season could be divided into two groups. Group 1 viruses (A/Aichi/4/92 and A/Aichi/7/92) agglutinated goose red blood cells (GRBC) and CRBC, while group 2 viruses (A/Aichi/24/92 and A/Aichi/26/92) did not agglutinate CRBC. There were no amino acid differences between them in the haemagglutinin (HA) polypeptide. Reassortment experiments between a group 1 virus (A/Aichi/4/92) or a group 2 virus (A/Aichi/24/92) and the A/WSN/33 influenza A (H1N1) virus strain suggested that the HA gene products of the viruses of both groups had lost the capacity to agglutinate CRBC. The HA proteins expressed on Cos cells by transfecting the cDNAs of the virus HA gene of A/Aichi/4/92 and A/Aichi/24/92 agglutinated GRBC but not CRBC. These experiments indicated that the HA proteins of H1N1 viruses of both groups isolated in 1992 had lost the ability to agglutinate CRBC even though the group 1 virions showed haemagglutinating capacity with CRBC. By using the cDNAs of the HA gene of seven natural isolates obtained from 1977 to 1992, it was found that the expressed HA proteins of influenza A (H1N1) viruses isolated since 1988 had lost the ability to agglutinate CRBC. Experiments with chimeric and point-mutated HA cDNAs of A/Aichi/24/92 showed that an amino acid change at residue 225, which occurred after 1986, and a cluster of amino acid changes at residues 193, 196 and 197, which occurred before 1986, were responsible for loss of the ability to agglutinate CRBC. Egg-adapted virus derived from A/Aichi/24/92 had one amino acid change at residue 225 compared to the parental virus.

Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro

ABSTRACT We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.