Atsuko Totsuka

Outbreak of Central Nervous System Disease Associated with Hand, Foot, and Mouth Disease in Japan during the Summer of 2000: Detection and Molecular Epidemiology of Enterovirus 71

Few outbreaks of the serious enterovirus 71 (EV71) infections, which affect the central nervous system (CNS), had been reported in Japan before 2000. During June through August 2000, a patient died of pulmonary edema caused by brainstem encephalitis accompanied by EV71‐induced hand, foot, and mouth disease (HFMD), and many patients complicated by serious CNS disease, including paralysis, were hospitalized in a restricted area in Hyogo Prefecture, Japan (K‐area). During the same period, endemics of HFMD were reported in other areas in Hyogo Prefecture, where EV71 was isolated from HFMD patients, but few patients developed aseptic meningitis. The isolations of EV71 from K‐area patients were difficult with the use of Vero cells, so the strains were isolated by use of GL37 cells; Vero cells, however, could isolate EV71 strains from other areas in Hyogo Prefecture. We sequenced VP4 coding regions of these EV71 isolates and found that the isolates from K‐area had the same sequence, which, except for one isolate, was different from the sequences of EV71 strains isolated from other areas of Hyogo Prefecture. Although these results were not enough to state that EV71 from K‐area was a virulent strain, it seemed reasonable to conclude that serious CNS diseases in K‐area were caused by EV71 because it was the only infectious agent detected in the inpatients of K‐area.

Shifting Seroepidemiology of Hepatitis A in Japan, 1973–2003

Background. Hepatitis A infection is caused by hepatitis A virus (HAV) contracted through fecal‐oral transmission. Life‐long immunity is conferred after infection. Improved sanitary conditions have generally resulted in a significant decline in the incidence of hepatitis A. However, a low incidence of infection results in increased HAV susceptibility. The present study investigates the prevalence of anti‐HAV antibody and clarifies the current HAV status and HAV susceptibility in Japan at 2003. Methods. A total of 2,430 serum specimens collected during 2003 from Japanese individuals ranging in age from 0–92 years, were tested for anti‐HAV antibody using an inhibition enzyme linked immunosorbent assay. All specimens were obtained from the WHO and the National Serum Reference Bank/National Institute of Infectious Diseases, Tokyo, Japan. Results. The overall seroprevalence was 12.2%. Anti‐HAV antibodies were rarely detected in individuals between 0–44 years of age. Starting from the age of 45–49 years, seropositivity gradually increased through age 65 years and above. Seroprevalence was not affected by gender, and geographic distribution did not affect age‐specific seroprevalence until the age of 60 years. Conclusions. HAV susceptibility in Japan is increasing annually. Particularly, the prevalence of anti‐HAV antibody in individuals older than 50 years in 2003 was 50.3%, which is significantly lower than that of corresponding studies in 1994 (74.3%), 1984 (96.9%) and 1973 (96.9%). The growing susceptible population of advanced age results in more frequent HAV infection among them. The surveillance of anti‐HAV antibody prevalence is useful for implementing preventive measures and for controlling the spread of HAV.

Hepatitis A Virus Proteins

The RNA genome of hepatitis A virus (HAV) shares common characteristics of the picornavirus family. However, the nucleotide or amino acid sequences are distantly related with other members of the family. Like other picornaviruses, HAV proteins are cleaved from a large polyprotein (PO), but the processing and some products are quite different. The 3C protein is the sole processing enzyme, and the primary cleavage takes place at the 2A/2B site. Several VP1-2A sites are proposed. In some strains, the intermediate VP1-2A polypeptides are assembled in the virion. The VP4 is very small and not detected in the mature virion. Some mutations in 2B, 2C and 3A proteins are identified to enhance viral replication or to induce cytopathogenic effects in the viruses adapted to cell cultures.

Hepatitis A Vaccine Development in Japan

In Japan, hepatitis A virus (HAV) infection has been infrequent for many years, with the result that a significant number of individuals have no immunity to the virus. Almost all people younger than 35 years of age have no antibody and are susceptible to HAV. This may cause large outbreaks if prophylactic measures are not available. In our efforts to develop a vaccine, a genotype 3B strain of HAV (KRM003) from a Japanese patient was isolated, adapted, and propagated in an African green monkey kidney (AGMK) cell culture. Formalin-inactivated virus preparation from this culture was highly immunogenic and protective in Saguinus monkeys. Employing a cloned virus (KRM003C3) as the seed, and an established AGMK cell line (GL37) as the cell substrate, we then developed for human use a new lyophilized inactivated vaccine containing neither adjuvant nor preservative. The pilot vaccine lots have been evaluated in 1869 volunteers as clinically tolerable and highly immunogenic.

Characterization of anti-idiotypic antibodies mimicking antibody- and receptor-binding sites on hepatitis A virus

Two anti-idiotypic monoclonal antibodies (mAb2s; named 94-2 and 94-7), were generated from a BALB/c mouse immunized with human monoclonal anti-hepatitis A virus (HAV) neutralizing antibody KF94. We characterized the properties of the mAb2s and determined interactions between mAb2s, KF94 and HAV using enzyme-linked immunosorbent assay, immunofluorescence assay and HAV infectivity assay. Inactivated HAV inhibited mAb2 binding to KF94, indicating that the mAb2s mimicked the HAV neutralization site that was complementary to the paratope of KF94. MAb2 94-7 competed with an anti-HAV cellular receptor antibody for binding to HAV-susceptible cells and partially blocked virus infection. We speculated that mAb2 94-7 mimicked a portion of the HAV receptor-binding site. The ability to generate mAb2 implies that HAV receptor-binding sites are exposed on the surface of HAV, permitting antibody access.

Hepatitis A virus strain KRM238 resistant at heat inactivation

Farcet and colleagues reported the significant difference in heat sensitivity of hepatitis A virus (HAV) variants during liquid heating of a human serum albumin preparation. Compared with a reference strain HM175 HAV stock, variants that were shown to differ in sequence at various singlenucleotide polymorphisms showed more heat resistance: approximately 3-log reduction in 25% albumin preparations compared with 5-log reduction of the reference HM175 strain, while 2-log reduction in 5% albumin preparations compared with 4-log reduction of the reference HM175 strain. These findings suggested that the differences in the exact strain of HAV used could affect the interpretation of virus inactivation capacity during manufacturing processes for biological medicines such as plasma products. HM175 strains belong to Genotype Ib. Shimasaki and colleagues also reported differences in heat sensitivity using Genotype IIIb HAV strains passaged in AGMK cell culture, KRM003G72 and KRM238G59. The KRM003G72 strain showed more than 5-log reduction after heating at 60°C for 10 hours in phosphate-buffered saline (PBS). In contrast, KRM238G59 showed only 2-log reduction under the same conditions. These strains were derived from fecal specimens by the sporadic case in Fukuoka, Japan, in 1979 and by an outbreak in Saga, Japan, in 1977, respectively. The original clinical viruses KRM003WT and KRM238WT were later proved to have the same nucleotide sequence from N50 to end. The infectious cDNA clone of heat sensitive KRM003G72 was replaced in the P1 region with the cDNA fragment from the KRM003WT. The recombinant virus with the wild-type capsid, FR1, rescued by transfection with the transcript RNA of the chimerical cDNA, showed heat-resistant kinetics similar to those of KRM238G59 (Figs. 1 and 2). Compared to wild-type sequence, KRM003G72 and KRM238G59 had three amino acid substitutions in P1 region: one amino acid substitution in a common residue as in VP3-94 (Glu to Asp) and two amino acid substitutions in different residues as in VP2-135 (Thr to HIs) and VP1-17 (Asn to Ser) and also in VP2-110 (Ile to Val) and VP1-237 (Asn to Asp). Further studies with recombinant virus showed that only VP1-17 (Asn to Ser) could be involved in heat sensitivity. These studies suggest a possibility that HAV strains in clinical materials may be more heat resistant than a certain population of laboratory strains adapted to cell cultures. Murozuka and colleagues evaluated heat inactivation of HAV KRM238 in 20 and 25% albumin preparations.