Kanetsu Sugawara

Effect of the Addition of Oligosaccharides on the Biological Activities and Antigenicity of Influenza A/H3N2 Virus Hemagglutinin

ABSTRACT Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.

Structural determination of gangliosides that bind to influenza A, B, and C viruses by an improved binding assay: Strain-specific receptor epitopes in sialo-sugar chains

An improved binding assay for detection of ganglioside receptors for influenza A, B, and C viruses was developed. In this system, the virions bound to gangliosides that were developed on a silica gel thin-layer plate were detected by mouse monoclonal antibody against viral hemagglutinin and peroxidase-conjugated anti-mouse immunoglobin. No hydrolysis of the gangliosides by viral receptor-destroying enzyme was detected in the present condition. The reactivity of the viruses to gangliosides depended on the amount of developed gangliosides (10 pmols-10 nmols), the molecular species of sialic acid, and their sugar sequences. Human influenza A (PR/8/34), B (Lee/40), and C (Ann Arbor/1/50) viruses bound different receptor epitopes of sialo-sugar chains of gangliosides. The A/PR/8 virus bound most effectively to Neu5Ac-containing lacto-series gangliosides carrying type I and type II sugar chains, followed by ganglio-series and hematoside-series gangliosides. The A/PR/8 virus weakly bound to Neu5Ac alpha 2,6lactotetraosylceramide [IV6(Neu5Ac)Lc4Cer] and Neu5Ac alpha 2,6paragloboside [IV6(Neu5Ac)nLc4Cer] carrying Neu5Ac alpha 2,6Gal sequence, although their Neu5Ac alpha 2,3Gal derivatives were the most potent gangliosides tested. B/Lee/40 bound restrictively to IV6(Neu5Ac)Lc4Cer and IV6(Neu5Ac)nLc4Cer, which carry Neu5Ac alpha 2,6Gal sequence, and type I and type II lacto-series sugar chain, respectively. C/Ann Arbor/1/50 reacted only with 9-O-Ac-Neu5Ac-carrying sugar chains in all the gangliosides tested. This method also allowed the microanalysis of receptor gangliosides of unknown samples. ESK cells, sensitive to the influenza A viruses infection, expressed several kinds of receptor active gangliosides, while those from ESK-R cells, resistant to the virus infection, were undetectable.

Antigenic structure of the haemagglutinin of human influenza A/H2N2 virus

The antigenic structure of influenza A/H2N2 virus haemagglutinin (HA) was analysed using 19 monoclonal antibodies (MAbs) against the HA of A/Kayano/57. The antibodies were classified into three groups: group I had both haemagglutination inhibition and neutralization activities, group II had neutralization activity but no haemagglutination inhibition activity and group III had neither activity. Analysis of escape mutants selected by each of the group I and II antibodies identified six distinct antigenic sites: four (I-A to I-D) were recognized by group I MAbs and two (II-A and II-B) were recognized by group II MAbs. Sequence analysis of the HA genes of the escape mutants demonstrated that sites I-A, I-B and I-C form a contiguous antigenic area that contains the regions corresponding to antigenic sites A, B and D on the H3 molecule and that sites I-D and II-B are the equivalents of sites E and C, respectively, suggesting that the antigenic structure of the H2 molecule is largely similar to that of the H3 molecule. However, the H2 molecule differed from the H3 molecule in having a highly conserved antigenic site (II-A) in the stem domain. It was also found that most of the escape mutants selected by antibodies to sites I-A, I-B and I-C acquired a new glycosylation site at position 160, 187 or 131, respectively, which indicates that A/H2N2 viruses have the potential to gain at least one additional oligosaccharide on the tip of the HA, although this has never occurred during 11 years of its circulation in humans.

Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007.

We isolated and identified six subgenogroups (B2, B4, B5, C1, C2, and C4) of enterovirus 71 (EV71) between 1990 and 2007 in Yamagata, Japan. We measured neutralizing antibody (NT Ab) titers against those subgenogroup strains and the BrCr reference strain for antigenic analysis. Serological analysis of 83 residents in Yamagata in 2004 showed that differences in the NT Ab titer of each individual against the different subgenogroups were mostly within 4-fold. Furthermore, sera from guinea pigs, immunized with the B2 and C1 strains indicated cross-antigenicity among the seven different subgenogroups. In conclusion, our results showed that cross-antigenicity exists among EV71 strains from different subgenogroups circulating in the community through genomic evolution. Our results also suggest that eliciting neutralizing antibodies against one genotype is likely to confer cross-neutralization against other genotypes.

Interspecies transmission of influenza C virus between humans and pigs

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule.

The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.

The synthesis of polypeptides in influenza C virus-infected cells.

The synthesis of virus-specific polypeptides was analyzed in MDCK cells infected with the JJ/50 strain of influenza C virus. In addition to three major structural proteins gp88, NP, and M, the synthesis of five polypeptides with molecular weights of 29,500 (C1), 27,500 (C2), 24,000 (C3), 19,000 (C4), and 14,000 (C5) was found in infected cells. None of these polypeptides were detected either in virions or in immunoprecipitates obtained after treatment of infected cell lysates with antiviral serum, suggesting that they are not viral structural proteins. Polypeptides C1-C5 were found to be synthesized in MDCK cells infected with different influenza C virus strains as well as in different host cell types infected with C/JJ/50. Further, it was observed that cellular protein synthesis was greatly reduced under hypertonic conditions, whereas the synthesis of C1-C5 was relatively unaffected. These results suggest that polypeptides C1-C5 are virus coded rather than host cell coded. Peptide mapping studies showed that each of polypeptides C3, C4, and C5 had a peptide composition similar to the M protein. The amount of C2 synthesized in infected cells was insufficient for mapping. This polypeptide was, however, found to rapidly disappear in pulse-chase experiments, suggesting that C2 is probably not unique but biosynthetically related to one of the other proteins. In contrast to these polypeptides, polypeptide C1 showed a map which is largely different from any major structural polypeptide. It therefore appears likely that C1 is a nonstructural protein of influenza C virus similar to the NS1 protein of influenza A and B viruses.

Antigenic and Genetic Characterization of Influenza C Viruses Which Caused Two Outbreaks in Yamagata City, Japan, in 1996 and 1998

ABSTRACT During the 3 years from January 1996 to December 1998, a total of 33 strains of influenza C virus were isolated from 10,726 throat swab specimens collected from children with acute respiratory illness who visited two pediatric clinics in Yamagata City, Japan. These 33 strains were isolated in clusters during two different periods, 20 strains in May to August 1996 and the remaining 13 in March to June 1998. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein and phylogenetic analysis of seven RNA segments showed that the 33 influenza C viruses isolated were antigenically and genetically similar and that they were reassortant viruses which had obtained PB2, PB1, HE, M, and NS genes from a C/pig/Beijing/115/81-like virus and P3 and NP genes from a C/Mississippi/80-like virus. These observations suggest strongly that during the survey period of 3 years, two outbreaks of influenza C occurred in Yamagata City, both of which were caused by a reassortant virus having the genome composition described above.

Antigenic and receptor binding properties of Enterovirus 68

ABSTRACT Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract. IMPORTANCE Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

Frequent Reassortment among Influenza C Viruses

ABSTRACT In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol. 81:1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.