Seiji Hongo

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.

Frequent Importation of Enterovirus 71 from Surrounding Countries into the Local Community of Yamagata, Japan, between 1998 and 2003

ABSTRACT Phylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel “B5.” Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.

Prolonged Norovirus Shedding in Infants 6 Months of Age With Gastroenteritis

Background: Noroviruses (NV) are one of the leading causes of gastroenteritis in young children; however, the duration of NV shedding in young children is not well known. Methods: Fecal specimens were collected from children with acute gastroenteritis at a pediatric clinic during the period from November to December 2002 and tested for NV by reverse transcription–polymerase chain reaction. Results: Of 71 children infected with NV, 60 (84.5%) were less than 3 years old. Among children aged <2 years and those aged 2 to 5 years, the duration of illness was longer (7 days versus 3.5 days, P = 0.0069), the maximum number of stools in a 24-hour period was greater (7 versus 3, P = 0.0078) and a 20-point severity score was higher (11 versus 8, P = 0.0031) in patients aged <2 years than in patients aged 2 to 5 years. Among the 23 children whose follow-up specimens were obtained, the median duration of NV shedding was 16 days (range, 5–47 days). Virus shedding for more than 2 weeks after onset was observed in 75% (6 of 8), 71.4% (5 of 7) and 25% (2 of 8) of children aged <1 year, 1 year and 2 to 3 years, respectively. Three infants aged ≤6 months continued to excrete NV for an extremely long period (more than 42, 44 and 47 days from onset) after recovery. Conclusion: Long-term virus shedding after the disappearance of clinical symptoms was observed. Caution should be exercised when handling the excrement of infants and young children infected with NV.

The Role of G-Protein-Coupled Receptor Kinase 5 in Pathogenesis of Sporadic Parkinson's Disease

Sporadic Parkinsons disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of α-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with α-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of α-synuclein at the plasma membrane and induced translocation of phosphorylated α-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of α-synuclein. Genetic association study revealed haplotypic association of the GRK5 gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of the GRK5 gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of α-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.

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.

Reactivation of Herpes Simplex Virus Type 1 and Varicella‐Zoster Virus and Therapeutic Effects of Combination Therapy With Prednisolone and Valacyclovir in Patients With Bell's Palsy

Objectives: To determine whether reactivation of herpes simplex virus (HSV) type 1 or varicella‐zoster virus (VZV) is the main cause of Bells palsy and whether antiviral drugs bring about recovery from Bells palsy.

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.

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.

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.