Akio Hagiwara

Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection.

Viruses isolated from patients with hand, foot and mouth disease in widespread outbreaks in Japan in 1973 were identified as enterovirus 71. Although cases with aseptic meningitis were observed concurrently, the main clinical symptom associated with enterovirus 71 infection was hand, foot and mouth disease.

Mutations in the 2C Region of Poliovirus Responsible for Altered Sensitivity to Benzimidazole Derivatives

ABSTRACT MRL-1237, [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl) methylbenzimidazole hydrochloride], is a potent and selective inhibitor of the replication of enteroviruses. To reveal the target molecule of MRL-1237 in viral replication, we selected spontaneous MRL-1237-resistant poliovirus mutants. Of 15 MRL-1237-resistant mutants obtained, 14 were cross-resistant to guanidine hydrochloride (mrgr), while 1 was susceptible (mrgs). Sequence analysis of the 2C region revealed that the 14mrgr mutants contained a single nucleotide substitution that altered an amino acid residue from Phe-164 to Tyr. Themrgs mutant, on the other hand, contained a substitution of Ile-120 to Val. Through the construction of a cDNA-derived mutant, we confirmed that the single mutation at Phe-164 was really responsible for the reduced susceptibility to MRL-1237. MRL-1237 inhibited poliovirus-specific RNA synthesis in HeLa cells infected with a wild strain but not with an F164Y mutant. We furthermore examined the effect of mutations of the 2C region on the drug sensitivity of cDNA-derived guanidine-resistant and -dependent mutants. Two guanidine-resistant mutants were cross-resistant to MRL-1237 but remained susceptible to another benzimidazole, enviroxime. Either MRL-1237 or guanidine stimulated the viral replication of two guanidine-dependent mutants, but enviroxime did not. These results indicate that MRL-1237, like guanidine, targets the 2C protein of poliovirus for its antiviral effect.

Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients.

SummaryWe examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5′ non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent than Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.

Genetic and phenotypic characteristics of enterovirus 71 isolates from patients with encephalitis and with hand, foot and mouth disease

SummaryBiological and biochemical characters of seven enterovirus 71 (E71) isolates were compared. Four isolates (two from patients with hand, foot and mouth disease [HFMD] and two from patients with encephalitis) grew in cynomolgus monkey kidney cells both at 39.5 and 35° C. However, the remaining three strains (from patients with HFMD) grew at 35° C, but not at 39.5° C. Three temperature-resistant and two temperature-sensitive strains were tested for neurovirulence in monkeys. Temperature-resistant strains were shown to be neurovirulent, whereas temperature-sensitive strains were less neurovirulent. The results suggest correlation between temperature-sensitive growth and neurovirulence in monkeys of E71. Variation in the electrophoretic mobility of the viral polypeptides was detected in three out of seven strains. The fingerprinting of oligonucleotides generated from the viral genome showed similar patterns in two isolates from patients with HFMD and one from patient with encephalitis and variable patterns in each genomic map of remaining four strains. These variations of polypeptide patterns and of oligonucleotide maps could not be correlated with pathogenicity (encephalitis or HFMD), temperature-sensitive growth and neurovirulence in monkeys.

Isolation of a temperature-sensitive strain of enterovirus 71 with reduced neurovirulence for monkeys.

Small- and large-plaque-producing viruses were selected from the enterovirus 71 (E71) prototype, BrCr. The small-plaque virus was a temperature-resistant (tr) strain which could multiply at 39.5 degrees C as well as 35 degrees C. The large-plaque virus was temperature-sensitive (ts) and could not grow at 39.5 degrees C. It was shown that the ts strain was much less neurovirulent for monkeys than the tr strain. Both tr and ts strains reacted with homologous and heterologous antibodies in cross-neutralization tests. While no difference was found between tr and ts strains in the size of virion polypeptides, the VP1 polypeptide could be differentiated by isoelectric focusing. Genomic differences between the two strains were found by oligonucleotide mapping.

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in Yokohama City between 1982 and 2000

Summary. Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980’s and 1990’s. Six EV71 isolates in the early 1980’s in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970’s. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan.

Common antigen between coxsackievirus A 16 and enterovirus 71.

Cross immunofluorescence revealed that coxsackievirus A 16 (CA 16) shared a common antigen with enterovirus 71 (E 71). The cross reactivity of these two serotypes was also examined by complement fixation test with purified virus preparations fractionated by sucrose density gradient centrifugation and two peaks of antigenicity were detected, one being type‐specific and the other cross‐reacting. The common antigen was heat‐stable and attributable to empty capsids. Immunodiffusion also revealed the common antigen. Infants without antibody to E 71 developed complement fixing and precipitin antibody to E 71 after recovery from hand, foot and mouth disease caused by CA 16.

Seroepidemiology of Enterovirus 71 among Healthy Children near Tokyo

In 1973 an epidemic of hand, foot and mouth disease (HFMD) took place throughout Japan (4). We reported that this epidemic was associated with enterovirus 71 (E71) infection, which had not been known to cause a wide-spread outbreak of the disease (1). In order to know whether or not E71 was prevalent in Japan before this epidemic, we carried out a sero-survey on neutralizing-antibody against E71 among healthy children. Sera of healthy children, from zero to ten years of age, were collected in Takatsu area, Kawasaki City near Tokyo, every March from 1966 to 1973. The

Two major strains of type 1 wild poliovirus circulating in Indochina.

Two hundred ninety-four isolates from 329 patients with acute flaccid paralysis in Cambodia and Vietnam during 1992-1995 were identified as type 1 wild polioviruses. Among these isolates, 85 were selected as geographic representatives and were examined by determining the nucleotide sequences of their genome in the VP1 region. The phylogenic analysis revealed that all of the isolates examined were classified into groups A and B. Isolates belonging to group A had been found only in northern Vietnam until 1993 but not in 1994 and 1995. Group B isolates were located in both northern and southern Vietnam and Cambodia. In 1994 and 1995, however, only group B isolates were found in the Mekong Delta area in southern Vietnam and Cambodia. Isolates of groups A and B were genetically different from strains previously isolated in other Asian countries. One of the two indigenous wild polioviruses still remains to be eliminated in this area.

Evaluation of an enzyme-linked immunosorbent assay based on binding inhibition for type-specific quantification of poliovirus neutralization-relevant antibodies.

To detect neutralization‐relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme‐linked immunosorbent assay (ELISA) based on monoclonal antibody‐binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti‐PV1 antibody, 98.3% (118/120) for anti‐PV2, and 96.5% (82/85) for anti‐PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally‐infected populations.