Ken Honzumi

Genetic diversity of enterovirus 71 associated with hand, foot, and mouth disease epidemics in Japan from 1983 to 2003

Background: Enterovirus 71 (EV71) is one of the major etiologic agents of hand, foot and mouth disease (HFMD). The surveillance data indicate that EV71 infection follows an epidemic mode of transmission, causing large outbreaks and then becoming quiescent for a few years. Methods: We investigated the genetic diversity of a total of 121 EV71 strains isolated from patients with HFMD in Fukushima, Japan, from 1983 to 2003 and compared their genetic relation with the 164 EV71 strains isolated in the world using phylogenetic analysis based on the VP4 sequence. Results: We observed EV71-related HFMD outbreaks in Fukushima in 1984, 1987, 1990, 1993, 1997, 2000 and 2003. Phylogenetic reconstruction of EV71 strains isolated in Fukushima demonstrated 8 genetically distinct clusters, including 6 subgroups previously designated as B-1, B-2 and 3, B-4, C-1, C-2, and C-3 and 2 subgroups newly designated as B-5 and C-4. Additional 2 indistinct clusters belonged to genogroup C and were named C-U1 and C-U2. Of those subgroups, B-1, C-U1, C-U2, C-2, B4, and C-4 and B-5 dominantly related to epidemics that occurred in the years 1984, 1987 and 1990, 1993, 1997, 2000 and 2003, respectively. EV71 strains derived from each outbreak in Fukushima formed a single cluster with those isolated during almost the same time period in other area of Japan and in other countries. Conclusions: Our results suggested that the repeated EV71 outbreaks might be the result of the worldwide transmission of the newly introduced genetically divergent EV71 strains.

Genetic Diversity of Coxsackievirus A16 Associated with Hand, Foot, and Mouth Disease Epidemics in Japan from 1983 to 2003

ABSTRACT To clarify the chronologic genetic diversity of coxsackievirus A16 (CV-A16) strains associated with hand, foot, and mouth disease (HFMD) epidemics in a restricted area and their genetic relation with those isolated in other areas, we investigated the genetic diversity of the 129 CV-A16 strains associated with HFMD epidemics in Fukushima, Japan, from 1983 to 2003, and compared their genetic relation to 49 CV-A16 strains isolated in other areas of Japan and in China by using phylogenetic analysis based on the VP4 sequences. Phylogenetic reconstruction of the CV-A16 strains isolated in Fukushima from 1983 to 2003 demonstrated three distinct genetically divergent clusters related to HFMD epidemics that occurred from 1984 to 1994 (including the 1985 and 1991 outbreaks), HFMD epidemics from 1987 to 1998 (including the 1988 and 1998 outbreaks), and HFMD epidemics from 1995 to 2003 (including the 1995 and 2002 outbreaks). CV-A16 strains isolated during each period in Fukushima formed a single cluster with those isolated during essentially the same time period in other areas of Japan and in China. Our results demonstrated that prevalent CV-A16 strains causing HFMD in Fukushima, Japan, genetically changed twice during 21 epidemics, and changes were also observed in the CV-A16 strains causing HFMD epidemics in other areas. We concluded that repeated outbreaks of CV-A16-related HFMD in Japan were caused, in part, by the introduction of genetically changed CV-A16 strains, which might be transmitted overseas.

Application of PCR for various neurotropic viruses on the diagnosis of viral meningitis.

BACKGROUND Epidemiological studies have indicated that the majority of cases of aseptic meningitis result from viral infections. However, specific viral pathogens for aseptic meningitis can be identified in only some cases even if consistent conventional diagnostic methodologies rare used. OBJECTIVES To clarify the etiological agents of aseptic meningitis by means of polymerase chain reaction (PCR) for various neurotropic viruses. STUDY DESIGN Cerebrospinal fluid (CSF) samples were collected from 73 children suspected of having meningitis from November 1991 to December 1994. The samples were examined for infectious viruses by cell culture and for viral genomes by PCR. RESULTS AND CONCLUSIONS Of 45 samples from patients diagnosed with aseptic meningitis, positive PCR results for enterovirus, mumps virus, cytomegalovirus, and varicella-zoster virus were obtained from respectively 25, 14, 1, and 1. Viral pathogens were thus identified in 41 (91.1%) of the 45 CSF samples. By the combination of PCR methods with conventional virological methods, the diagnosis of viral meningitis was established in 97.8% of the 45 cases. Our findings prove that the application of PCR methods is useful for etiological study of aseptic meningitis, and that the vast majority of cases of aseptic meningitis result from viral infection.

Diagnosis of group A coxsackieviral infection using polymerase chain reaction

Aims: To examine the relation between enteroviral infection, especially group A coxsackieviral infection, and acute febrile illness over two summers using tissue culture and polymerase chain reaction (PCR). Methods: Throat swabs were collected from 246 children from June to August 1997 and 1998. Results: Enteroviruses were isolated from 33/246 samples and 35 other viruses were isolated. Enteroviral genomes were detected in 54/178 samples from which no virus was isolated. Of 41 enteroviral genotypes identified by sequence analysis of PCR products, 38 were group A coxsackieviruses, which are usually difficult to isolate using tissue culture. Conclusion: Results indicate that viral detection and identification based on PCR is useful in the diagnosis of group A coxsackieviral infection.

Application of polymerase chain reaction and subsequent phylogenetic analysis to the diagnosis of enteroviral infection in the central nervous system

BACKGROUND Enteroviral infections of the central nervous system (CNS) are often difficult to diagnose, even if consistent conventional laboratory methodologies are used. OBJECTIVES To clarify the efficiency of two polymerase chain reaction (PCR) methods for the sensitive detection of enteroviruses and for the identification of enteroviral genotypes based on phylogenetic analysis of the amplified genome sequences, and to facilitate the diagnosis of enteroviral infection in CNS. STUDY DESIGN Cerebrospinal fluid (CSF), throat swab, rectal swab, and/or serum samples were collected from 171 patients with aseptic meningitis and 67 patients with febrile seizures. The samples were tested for the presence of enteroviruses by cell culture and PCR methods for the detection and identification of enteroviruses. RESULTS In 111 (64.9%) of 171 patients with aseptic meningitis, enteroviruses were isolated by cell cultures from any site. In 143 (83.6%) patients, including 110 of 111 patients with aseptic meningitis, the enteroviral genome was detected in CSF by PCR. No enterovirus was isolated from any site for the 67 patients with febrile seizures. PCR detected the enteroviral genome in CSF samples from 13 (61.9%) of 21 patients who developed febrile seizures in the summer (June-August). Phylogenetic analysis of amplified genome sequences showed that the major pathogens of febrile seizures in summer were group A coxsackieviruses, which are usually difficult to isolate by cell culture. CONCLUSION PCR methods for the detection and identification of enteroviruses were useful for the diagnosis of enteroviral infection in CNS.

Quantitative analysis of influenza A (H3N2) E119V and R292K variants in clinical specimens by real-time reverse transcription polymerase chain reaction.

BACKGROUND Because influenza virus isolates after cell culture are required to determine their susceptibility to neuraminidase inhibitors, the differences in normal or low-susceptibility variant population frequencies between clinical samples and isolates have not been considered. OBJECTIVES To identify variations in low-susceptibility populations in clinical samples after initiation of oseltamivir and zanamivir therapy by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). STUDY DESIGN We measured the populations of the low-susceptibility influenza A H3N2 variants E119V and R292K by qRT-PCR using 305 nasal aspiration samples collected over time from 13, 16, and 11 patients treated with no neuraminidase inhibitors, oseltamivir, and zanamivir, respectively. The variant population in the isolates was also determined when the population of low-susceptibility variants in the clinical samples increased following treatment. Moreover, the susceptibility of all isolates was measured. RESULTS The E119V variant was detected in only one patient during oseltamivir therapy, exhibiting decreased susceptibility to oseltamivir. Prior to treatment, R292K variants were detected in all clinical samples; however, they comprised only a small fraction of the total population. The proportion of the R292K variant in clinical samples increased for 6/27 (22.2%) patients treated with oseltamivir or zanamivir, whereas an increase in the proportion of the R292K variant in virus isolates was observed in only one patient. CONCLUSIONS Discrepancies in the proportion of R292K variants between clinical samples and isolates should be suspected in clinical settings. qRT-PCR is useful for quantitative analysis of drug-resistant influenza virus and for immediate notification of the result.