Teruo Yamashita

Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.

Detection of human parechoviruses from clinical stool samples in Aichi, Japan.

ABSTRACT Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5′ untranslated region (5′UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, “hand, foot, and mouth disease,” aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.

Hepatitis A, B and C seroprevalence in Pakistan

A cross sectional study was conducted to determine the seroprevalence of Hepatitis A, B, and C virus in healthy Pakistani children. HAV IgG antibody was assayed in 258 subjects and it was found that 94% children by 5 years of age had HAV IgG-antibody. The overall seroprevalence of HAV IgG antibody was 55.8% and IgM 5.3%. HBVsAb levels assayed in 236 healthy children showed a seroprevalence of 2.97%. Similarly, HCV antibody seroprevalence was found to be a low 0.44% in healthy children. HAV is a major cause of Hepatitis, as compared to HBV and HCV which are of low endemicity.

Identification of Aichi Virus Infection by Measurement of Immunoglobulin Responses in an Enzyme-Linked Immunosorbent Assay

ABSTRACT Using inhibitory enzyme-linked immunosorbent assay, seroconversions to Aichi virus were detected in 24 (42.9%) of 56 patients with gastroenteritis in six outbreaks. Virus-specific immunoglobulin M (IgM) was detected in convalescent-phase sera from 7 of 24 patients. Of the other 17 patients, 12 developed a significant increase in both IgA and IgG levels and 5 developed a significant increase in IgG alone.

Molecular identification of enteroviruses including two new types (EV-98 and EV-107) isolated from Japanese travellers from Asian countries.

Of 58 enterovirus strains isolated from Japanese travellers returning from Asian countries, eight were non-serotypable with existing antisera. By sequencing a part of the VP1 region, six of these strains were typed as echovirus 9, enterovirus (EV)-73, EV-79 or EV-97. The nucleotide identity of the VP1 region of isolate T92-1499 to all enterovirus prototypes was <70 %. The VP1 sequence of isolate TN94-0349 was closely related to coxsackievirus (CV)-A9 (73.3 % nucleotide identity), but the virus could not be neutralized with a serum raised against the prototype CV-A9 strain. On the basis of complete molecular comparisons, T92-1499 and TN94-0349 were identified as EV-98 and EV-107, respectively, by the ICTV Picornavirus Study Group. Serum neutralization tests of Japanese individuals revealed a seroprevalence rate of 11 % for EV-73, and even lower seroprevalence rates, 1.0-3.8 %, were found for the other new enteroviruses, suggesting that prior circulation of these viruses in Japan was unlikely.

Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan

Four strains of Orientia tsutsugamushi (KN‐1, KN‐2, KN‐3 and GJ‐1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN‐1 (identified as Kawasaki type), GJ‐1 (identified as Kuroki type) and KN‐2 strains were found to be non‐lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (106 × being the 50% mouse infectious dose). On the other hand, the KN‐3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN‐1 and KN‐2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN‐3, Karp and Kato strains. After infection with the GJ‐1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.

Enteric viral infections in pre-school children in Karachi, Pakistan.

A prospective study was conducted in Karachi, Pakistan on the virology of enteropathogens excreted by children with acute gastroenteritis and the results were compared with a control group of healthy children. Rotavirus and Adenovirus detection was done using ELISA techniques, while enterovirus isolation was done by virus culture. In 1990, 12.3% children with acute watery diarrhoea excreted rotavirus, as compared to 24.4% children in 1991. None of the healthy children excreted adenovirus 40 and 41. Preliminary results of 1992 revealed that rotavirus was seen in 13% of children with acute watery diarrhoea and adenovirus in 10% of children. Enteroviruses were isolated in the same frequency in all three groups i.e. children with acute watery diarrhoea, children with poliomyelitis and healthy children. Non-polio enteroviruses were excreted in 50–52% in all the 3 groups. The rate of enterovirus excretion is much higher than seen in other developed countries and is the same in children with diarrhoea and healthy children.

VI, 3. Molecular biology and epidemiology of Aichi virus and other diarrhoeogenic enteroviruses

Publisher Summary The virion of the Aichi virus contains a single-stranded RNA molecule as the genome. The homology of Aichi virus structural proteins (VP0, VP3, and VP1) with corresponding polypeptides of other picornaviruses varies between 19% and 32%. The epidemiology of the Aichi virus as a medically important pathogen has not been well defined. Stool samples from adult patients in six oyster-associated gastroenteritis outbreaks were examined for variation, based on their reactivity with a monoclonal antibody raised against the standard strain (A486/88) and on reverse transcription–polymerase chain reactions (RT-PCR) of three genomic regions. Antibody to the Aichi virus could be detected using a neutralization test and an enzyme-linked immunosorbent assay (ELISA). These methods were used for the identification of Aichi virus infection in paired serum samples. The chapter concludes with a discussion on other diarrheagenic enteroviruses.

Virological and serological studies on poliomyelitis in Karachi, Pakistan. I. Outbreaks in 1990-91.

Between October 1989 and September 1991, 124 cases of poliomyelitis visited the Department of Paediatrics, Civil Hospital Karachi, Pakistan. The majority of them were between 6 months and 2 years of age and the epidemics occurred during the hot seasons. The dominant serotype was poliovirus type 1 during the epidemic season in 1990 and type 2 in 1991. All the polioviruses isolated from the patients were wild‐type. Virological studies also disclosed that enteroviruses other than polioviruses were prevalent among healthy children as well as among diarrheal and polio patients.

Case-based surveillance enhanced with measles virus detection/genotyping is essential to maintain measles elimination in Aichi Prefecture, Japan

BACKGROUND Japan was verified as having achieved measles elimination by the Measles Regional Verification Commission in the Western Pacific Region in March 2015. Verification of measles elimination implies the absence of continuous endemic transmission. After the last epidemic in 2007 with an estimated 18,000 cases, Japan introduced nationwide case-based measles surveillance in January 2008. Laboratory diagnosis for all suspected measles cases is essentially required by law, and virus detection tests are mostly performed by municipal public health institutes. Despite relatively high vaccination coverage and vigorous response to every case by the local health center staff, outbreak of measles is repeatedly observed in Aichi Prefecture, Japan. METHODS Measles virus N and H gene detection by nested double RT-PCR was performed with all specimens collected from suspected cases and transferred to our institute. Genotyping and further molecular epidemiological analyses were performed with the direct nucleotide sequence data of appropriate PCR products. RESULTS Between 2010 and 2014, specimens from 389 patients suspected for measles were tested in our institute. Genotypes D9, D8, H1 and B3 were detected. Further molecular epidemiological analyses were helpful to establish links between patients, and sometimes useful to discriminate one outbreak from another. All virus-positive cases, including 49 cases involved in three outbreaks without any obvious epidemiological link with importation, were considered as import-related based on the nucleotide sequence information. Chain of transmission in the latest outbreak in 2014 terminated after the third generations, much earlier than the 2010-11 outbreak (6th generations). CONCLUSION Since 2010, almost all measles cases reported in Aichi Prefecture are either import or import-related, based primarily on genotypes and nucleotide sequences of measles virus detected. In addition, genotyping and molecular epidemiological analyses are indispensable to prove the interruption of endemic transmission when the importations of measles are repeatedly observed.