Setsuko Nakajima

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.

Seroprevalence of Noroviruses in Swine

ABSTRACT Noroviruses (NVs) are important human pathogens that cause acute gastroenteritis. Genetically related animal enteric NVs have also been described, but there is no evidence of interspecies transmission of NVs. In this study we characterized antibody prevalence among domestic pigs by using recombinant capsid antigens of two human NVs (Norwalk and Hawaii) and one swine NV (SW918) that is genetically related to GII human NVs. Recombinant SW918 capsid protein expressed in baculovirus self-assembled into virus-like particles (VLPs) that were detected by antibodies against GII (Hawaii and Mexico), but not GI (Norwalk and VA115), human NVs. NVs recognize human histo-blood group antigens as receptors, but SW918 VLPs did not bind to human saliva samples with major histo-blood group types. Seventy-eight of 110 (71%) pig serum samples from the United States and 95 of 266 (36%) pig serum samples from Japan possessed antibodies against SW918. Serum samples from pigs in the United States were also tested for antibodies against human NVs; 63% were positive for Norwalk virus (GI) and 52% for Hawaii virus (GII). These results indicate that NV infections are common among domestic pigs; the finding of antigenic relationships between SW918 and human NVs and the detection of antibodies against both GI and GII human NVs in domestic animals highlights the importance of further studies on NV gastroenteritis as a possible zoonotic disease.

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.

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.

Identification of the binding sites to monoclonal antibodies on A/USSR/90/77 (H1N1) hemagglutinin and their involvement in antigenic drift in H1N1 influenza viruses.

We have determined nucleotide sequences of the HA1 portion of the hemagglutinin (HA) gene of the parental A/USSR/90/70 (H1N1) virus and its eight variants selected in vitro with six monoclonal antibodies to study antigenic determinants. The HA1 gene of one of the variants (B-1-23) was cloned in bacteria and its nucleotide sequence was determined by the Maxam-Gilbert method. The nucleotide sequence of the variant was confirmed by the dideoxy chain termination method. The gene sequences of the other viruses were determined by the latter method. Three variants with reduced reactivity in HI test only with the selecting antibodies possessed one amino acid substitution. On the other hand, most other variants which had the changed reactivity to multiple antibodies in HI test possessed more than one substitution. Comparison of the amino acid sequences of the HA1 molecule, deduced from the nucleotide sequences, suggested that the monoclonal antibodies W18 and 264 reacted with epitopes located on the area involving amino acid residues 125C and 189-190, respectively, whereas, the antibodies 22 and 70 reacted with epitopes involving amino acid residues 129, 132, and 157. The epitope recognized by antibody 110 overlapped with that of W18, and the epitope recognized by antibody 385 was located on the area involving at least amino acid residues 129, 159, and 189, which overlapped with some of the above epitopes. The sequence analysis with B-1-23 variant selected with antibody 264 clearly showed that in A/USSR/77 viruses, a single substitution at amino acid residue 190 effectively changes the epitope and caused a significant antigenic variation detectable by postinfection ferret sera.

Amino-acid change on the antigenic region B1 of H3 haemagglutinin may be a trigger for the emergence of drift strain of influenza A virus

Sera from 27 children and eight older persons, which had been collected in 1998 and 1999 and showed haemagglutination-inhibition (HI) activity against influenza A/Sydney/5/97 (H3N2) strain, were characterized with a binding assay using chimeric haemagglutinin (HA) proteins between A/Aichi/2/68 (A/AI/68) and A/Sydney/5/97 (A/SD/97) strains. Sera from the young children had a tendency to recognize only the antigenic site B1 of the HA1 region. On the other hand, sera of the older individuals were fully reactive to all antigenic sites of HA1 except antigenic site D. Recent epidemic strains, A/Panama/2007/99 (A/PM/99)-like viruses have differences in amino acids in antigenic sites A, C, and B2 but not B1. However, human antisera obtained even from young children had HI activity to Panama-like viruses. The limited epidemic of A/PM/99-like viruses may have been due to the existence of antibody against B1, which had been produced in response to infection by the A/SD/97-like viruses.

Further genetic evidence for maintenance of early Hong Kong-like influenza A(H3N2) strains in swine until 1976

Abstract The oligonucleotide map of the whole RNA of A/swine/Hong Kong/3/76 (H3N2) virus showed a relationship to, but considerable differences from, the maps of human isolates of influenza A(H3N2) with which this isolate antigenically cross-reacted. Comparison of the individual oligonucleotide spots of whole virus RNAs confirmed previous results of antigenic analysis that A/swine/Hong Kong/3/76 virus was more similar to an early A/Hong Kong/68 virus than to later H3N2 viruses which circulated in man during 1976. On the other hand, another swine isolate, A/swine/Hong Kong/4/76, showed a quite similar oligonucleotide map to that of the contemporary prevalent A/Victoria/75-like viruses to which this strain was reported to be antigenically similar. Comparison of oligonucleotide maps of individual RNA segments indicated that all genes of the A/swine/Hong Kong/3/76 virus were derived from a human H3N2 virus. The findings provide biochemical evidence that A/swine/Hong Kong/3/76 virus represents a 1968 Hong Kong-like virus that underwent genetic mutation without extensive change of its antigenicity during maintenance in the swine population.