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Purpose. The aim of the study was isolation of adenoviruses by cell culture and identification using polymerase chain reaction (PCR) and phylogenetic analyses in patients clinically diagnosed with viral conjunctivitis in Ankara, Turkey. Methods. Conjunctival swabs from 34 patients with acute conjunctivitis were tested using cell culture isolation and PCR for adenovirus detection. PCR-positive samples were sequenced and typed. Results. The positive results of adenovirus were 26.5% (9 of 34) by the PCR method and 20.6% by culture isolation. Nine samples positive at PCR were identified by phylogenetic analyses as human adenovirus 8 (HAdV-8) (4 of 9), HAdV-3 (3 of 9), HAdV-4 (1 of 9), and HAdV-B (1 of 9). Conclusions. Our study showed types of adenoviruses in patients with ocular infection that occurred in this region of Turkey for the first time. Furthermore, sequence-based typing method is an efficient, accurate, and rapid means of diagnosis and typing of the adenovirus and has significant clinical and epidemiologic implications. HAdV-8 was major type for acute conjunctivitis in Ankara, Turkey. Further studies are required to reveal the major types of HAdVs that cause ocular diseases in this region of the world.

The VP4, VP2, and VP1 gene regions were evaluated for their usefulness in typing human enteroviruses. Three published RT-PCR primers sets targeting separately these three gene regions were used. Initially, from a total of 86 field isolates (36 HEV-A, 40 HEV-B, and 10 HEV-C) tested, 100% concordance in HEV-A was identified from all three gene regions (VP4, VP2, and VP1). However, for HEV-B and HEV-C viruses, only the VP2 and VP1 regions, and not VP4, showed 100% concordance in typing these viruses. To evaluate further the usefulness of VP4 in typing HEV-A enteroviruses, 55 Japanese and 203 published paired VP4 and VP1 nucleotide sequences were also examined. In each case, typing by VP4 was 100% in concordance with typing using VP1. Given these results, it is proposed that for HEV-A enteroviruses, all three gene regions (VP4, VP2, and VP1), would be useful for typing these viruses. These options would enhance the capability of laboratories in identifying these viruses and would greatly help in outbreaks of hand, foot, and mouth disease.

Human adenovirus type 8 (HAdV-8) and 37 (HAdV-37) cause epidemic keratoconjunctivitis (EKC) associated with community-acquired and nosocomial infections. The nucleotide sequences of the entire hexon and fiber genes of eight HAdV-8 and 26 HAdV-37 strains were analysed and the transition mutations in each gene were compared among strains. Compared with prototype strains, the hexon gene of HAdV-8 and -37 strains showed between two and seven and one and twelve variations at nine and 21 different positions, respectively. All of these, except one position in HAdV-37, were located in the conserved region 4 (C4). There were only three polymorphisms in the fiber gene of both HAdV-8 and HAdV-37, fewer than those in C4. The nucleotide sequence of HAdV-8 and -37 C4 might be readily modified during EKC epidemics.

In 2000, we encountered cases of nosocomial infections with epidemic keratoconjunctivitis (EKC) at a university hospital in Kobe, in the western part of Japan. Two human adenovirus (HAdV) strains, Kobe-H and Kobe-S, were isolated from patients with nosocomial EKC infection. They were untypeable by existing neutralizing antisera; however, the isolate was neutralized with homologous antisera. We then encountered several cases of EKC due to nosocomial infections in eye clinics in different parts of Japan. A total of 80 HAdVs were isolated from patients with EKC at eight different hospitals. The partial hexon gene sequences of the isolates were determined and compared to those of the prototype strains of 51 serotypes. All isolates had identical partial hexon nucleotide sequences. Phylogenetic analysis classified these isolates into species of HAdV-D. The isolates showed 93.9 to 96.7% nucleotide identity with HAdV-D prototype strains, while all 32 HAdV-D prototype strains ranged from 93.2 to 99.2% identity. The sequences of the loop 2 and fiber knob regions from the representative strain, Kobe-H, were dissimilar in all prototype strains of 51 serotypes. We believe that this virus is a novel serotype of HAdV that causes EKC.

In Neisseria gonorrhoeae , the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava , has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae , and its sensitivity was ≥10 1 copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of ≥0.5 μg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.

A lateral-flow immunochromatography (IC) assay for the detection of human metapneumovirus (hMPV) has been developed by using two mouse monoclonal antibodies to the nucleocapsid protein of hMPV. The purpose of this study was to compare the virus detection rate in nasopharyngeal secretions by the IC assay with that by real-time reverse transcription-PCR (RT-PCR). We collected nasopharyngeal swab samples from 247 children with respiratory symptoms in Sapporo, Japan, during the period from April to July 2007. Sixty-eight of the 247 children were positive for hMPV by real-time RT-PCR. When the real-time RT-PCR was used as the reference standard, the IC assay results were positive for 48 of the 68 real-time RT-PCR-positive children (70.6% sensitivity) and 8 of the 179 real-time RT-PCR-negative children (95.5% specificity). Although the sensitivity of the IC assay is lower than that of real-time RT-PCR, the IC assay is a rapid and useful test for the diagnosis of hMPV infections in children.

Background: The aim of the present paper was to investigate the molecular epidemiology of norovirus gastroenteritis in Japan using polymerase chain reaction (PCR) and subsequent phylogenetic analysis. Methods: From September 2001 to August 2003, 515 stool samples or rectal swabs were collected from almost all children visiting the Department of Pediatrics, Public Soma General Hospital with gastroenteritis. Samples were examined on reverse transcription (RT)-PCR to detect norovirus genome. The nucleotide sequences of the PCR products were determined and phylogenetic analysis performed. Results: The norovirus genome was detected in 66 samples. The peak season of norovirus gastroenteritis was from November 2001 to February 2002 and from September 2002 to December 2002. Norovirus gastroenteritis occurred most frequently in 1-year-old children. Norovirus strains produced four distinct clusters on phylogenetic analysis. Some strains detected in Soma were closely related to the strains detected in other regions in the world. The Mexico type and Lordsdale type were predominant in the 2001/2002 and 2002/2003 seasons, respectively, and the outbreaks continued for several months. Conclusions: Genetically different noroviruses might cause repeated gastroenteritis outbreaks every year in the Soma area. The long duration of the outbreak by a predominant strain in an epidemic season and the prevalence of infection mainly in the young age group suggested that norovirus epidemics were caused by person-to-person transmission rather than foodborne transmission. Based on molecular epidemiology, it is suggested that the annual prevalence of norovirus gastroenteritis in the Soma area might be caused by person-to-person transmission of genetically different norovirus strains, which might be transmitted from other region in the world.

The identification and geographic distribution of the herpes simplex virus type 1 (HSV-1) BglII restriction fragment length polymorphism (RFLP) variants named BgKLand BgOLin clinical isolates from orolabial and cutaneous sites were described in our previous reports, in which the dispersion and replacement of HSV-1 variants were proposed. The base substitution sites deduced from the BgKLmultiple RFLP variations were mapped to the UL12 (DNase), RL2 (α0 transactivator), and latency-associated transcript genes in the present study. The results show that the relative frequencies (RFs) of BgKLare significantly higher in orolabial and cutaneous HSV-1 infections than in ocular infections. For the BgOLvariant, the opposite was found; i.e., the RF of BgOLwas significantly lower in orolabial and cutaneous infections than in ocular infections. No significant differences in the RFs of non-BgKL:non-BgOLisolates were observed. The ratio of the BgKLRF to the BgOLRF was much higher for the orolabial and cutaneous infection groups than for the ocular infection group, whereas the BgKLRF-to-non-BgKL:non-BgOLRF ratios for the former groups were slightly higher than those for the latter group. The higher efficiency of orolabial and cutaneous infections caused by BgKLcompared to the efficiency of infections caused by BgOLallows BgKLto spread more efficiently in human populations and to displace BgOL, because the mouth and lips are the most common HSV-1 infection sites in children. The present study supports our HSV-1 dispersion-and-replacement hypothesis and suggests that HSV-1, the latency-reactivation of which allows variants to accumulate in human populations, has evolved under competitive conditions, providing a new perspective on the polymorphism or variation of HSV-1.

We previously reported a significant association between Ureaplasma urealyticum (biovar 2) and nongonococcal urethritis (NGU). We also found that the presence of Ureaplasma parvum (biovar 1) in the male urethra might be the result of colonization.The objective of this study was to clarify the pathogenic role of human Ureaplasma in NGU by assessing the association of bacterial loads with clinical findings and inflammatory responses in the urethra.The 16S rRNA gene of Ureaplasma was quantified by a TaqMan-based real-time polymerase chain reaction assay in first-pass urine from 37 men with Ureaplasma-positive nonmycoplasmal nonchlamydial NGU (NMNCNGU) and 30 Ureaplasma-positive men without urethritis.U. urealyticum (biovar 2) loads in 23 men with NMNCNGU were significantly higher than those in 14 men without urethritis. However, U. parvum (biovar 1) loads did not differ significantly between 14 men with NMNCNGU and 20 men without urethritis.The association of increased U. urealyticum (biovar 2) loads with symptomatic urethritis suggests that U. urealyticum (biovar 2) may be a pathogen of NGU. Our results also suggest that the presence of U. parvum (biovar 1) may not be significant in the development of NGU.

Objectives In Neisseria gonorrhoeae, the mosaic structure of penicillin-binding protein 2 (PBP 2), composed of fragments of PBP 2 from Neisseria cinerea and Neisseria perflava, was significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to determine the affinity of mosaic PBP 2 for cephalosporins in N. gonorrhoeae. Methods Two types of non-mosaic PBP 2 from the type strain of N. gonorrhoeae (ATCC 19424) and a clinical strain (GU01-29), as well as the mosaic PBP 2 from a clinical strain (GU01-89), were expressed in insect cells, and recombinant PBP 2s were purified. ATCC 19424 and GU01-29 were susceptible to cephalosporins. GU01-89 showed decreased susceptibility to cephalosporins. Bindings of fluorescent penicillin to PBP 2 were characterized by the Scatchard plot analysis. The affinity of the recombinant PBP 2s for cefdinir, cefixime and ceftriaxone was determined by PBP 2 competition assays with fluorescent penicillin. Results The K(d) value of mosaic PBP 2 for fluorescent penicillin was higher than that of non-mosaic PBP 2s. The affinity of mosaic PBP 2 for cefdinir or cefixime was lower than that of the non-mosaic PBP 2s. The affinity of the mosaic PBP 2 for ceftriaxone was not changed, compared with that of the non-mosaic PBP 2s. Conclusions Other mechanisms may be involved in clinical isolates with decreased susceptibility to cephalosporins, but this study suggests that the decreased affinity of mosaic-structure recombinant PBP 2 for oral cephalosporins may contribute to decreased susceptibility to these antibiotics in N. gonorrhoeae.

Human metapneumovirus (hMPV) nucleocapsid (N) protein is a major structural protein that encapsidates the RNA genome and is essential for replication and transcription of the hMPV genome. We developed two mouse monoclonal antibodies (MAbs), designated 3D1 and 5B10, against N protein of hMPV and characterized them by an immunofluorescence assay and an immunoprecipitation assay using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV N protein. Both MAbs were found to be reactive to two groups of hMPV by an immunofluorescence assay using two groups of hMPV-infected cells. A chromatographic immunoassay (lateral flow assay) was developed using the MAbs. The assay is a sandwich immunoassay that uses a paper membrane with a gold colloid-conjugated MAb (5B10) in a liquid phase and an MAb (3D1) in a solid phase. We preliminarily examined the sensitivity and specificity using hMPV-infected cells, Tn5 insect cells infected with a recombinant baculovirus-expressing hMPV N protein, hMPV, and purified N protein. The assay had good specificity and sufficient sensitivity to detect hMPV. Therefore, the assay may be a rapid and useful test for diagnosis of hMPV infections.

Around one million people are affected by adenoviral keratoconjunctivitis a year in Japan, and it is recognized as one of the major pathogens of ophthalmological nosocomial infection worldwide. Although cidofovir can be used systemically for immunocompromised patients with disseminated adenoviral infection, no specific anti-adenoviral agent has been established for the treatment of adenoviral infection. We evaluated the anti-adenoviral effect of anti-HIV (human immunodeficiency virus) agents in this study. Five anti-HIV agents (zalcitabine, stavudine, nevirapine, indinavir and amprenavir) were subjected to in vitro evaluation. A549 cells were used for viral cell culture, and adenovirus serotypes 3, 4, 8, 19 and 37 were used. After calculating CC50 (50% cytotoxic concentration) of each agent by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we cultured adenovirus with the agents for seven days and quantitatively measured extracted adenoviral DNA by real-time PCR. Among the anti-HIV drugs, zalcitabine and stavudine, both nucleoside reverse transcriptase inhibitors, showed significant anti-adenoviral activity. In contrast, nevirapine, a non-nucleoside reverse transcriptase inhibitor, and indinavir and amprenavir, which are both protease inhibitors, were ineffective against adenovirus. These results indicate that zalcitabine and stavudine are possible candidates for the local and systemic treatment of adenoviral infection, and the anti-adenoviral effect might depend on the pharmacological properties of anti-HIV agents. The chemical properties on the clinical safety for systemic and local application need to be determined in order to for these drugs to be accepted for the treatment of adenovirus in clinical settings.

Background/Aims: In adenoviral conjunctivitis, the infection process starts by the attachment of adenoviral fibers to conjunctival epithelial cells that contain the receptor for the adenovirus. The α5β1 integrin receptor ligand, GRGDSP peptide, contains the arginine-glycine-aspartate-binding motif which is common to the Coxsackie adenovirus receptor and integrins that are known to be adenoviral receptors. We evaluated the antiadenoviral effect of an expected adenoviral receptor inhibitor, GRGDSP peptide,in vitro. Methods: Adenovirus types 3, 4, 8, 19 and 37 were used. After calculating the 50% cytotoxic concentration of GRGDSP peptide, the adenovirus was cultivated with the agent for 7 days under serial dilution. Adenoviral DNA was qualitatively measured by real-time PCR. Results: GRGDSP peptide showed an inhibitory effect against adenoviral proliferation in all serotypes except type 4 in a dose-dependent manner. Conclusion: This result suggests that the α5β1 integrin receptor ligand, GRGDSP peptide, has antiadenoviral activity in vitro, and the possibility of being used for local treatment of epidemic keratoconjunctivitis.

Detection of antibodies against individual proteins of human metapneumovirus (hMPV) is important in the analysis of immune responses to hMPV. Specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The results were compared with those of immunofluorescence assays (IFAs) based on hMPV-infected LLC-MK2 cells, which expressed the whole hMPV proteins. Thirty (61.2%) and 31 (63.3%) of 49 serum samples with titers of >/=1:160 by IFA reacted with N and M proteins, respectively. Only 2 (4.2%) of 11 serum samples with titers of 1:80 by IFA reacted with N and M proteins. Antibodies against N and M proteins were not detected in 37 serum samples with titers of

Human metapneumovirus (hMPV) F protein promotes fusion of viral and cell membranes, and is thought to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. In this paper, the development of two mouse monoclonal antibodies (MAbs) by immunization with hMPVinfected cells is described. Immunofluorescence assay (IFA) using hMPV F protein-expressing cells indicated that two MAbs, designated 1G3 and 9B10, recognized hMPV F protein. Both MAbs were found to be reactive to two groups of hMPV by an IFA using two groups of hMPV-infected cells. The 9B10 mAb had strong neutralizing activity against both groups of hMPV, while the 1G3 MAb had only weak neutralization activity. These results indicate that the hMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Production of MAbs to the hMPV F protein is critical for development of diagnostic techniques, vaccine research, and studies on viral pathogenesis.

Background: Our previous study suggested a significant association between Ureaplasma urealyticum and nongonococcal urethritis (NGU). However, association of the serovars of U. urealyticum with NGU remains unclear. A polymerase chain reaction (PCR)-based assay can distinguish 4 serovars of Ureaplasma parvum from each other and categorize 10 serovars of U. urealyticum into 3 subtypes: subtype 1 (serovars 2, 5, 8, and 9), subtype 2 (serovars 4, 10, 12, and 13), and subtype 3 (serovars 7 and 11). Goal: The goal of this study was to determine which subtypes of U. urealyticum are associated with NGU as determined by PCR-based assay. Study: The prevalence of U. urealyticum subtypes in 106 ureaplasma-positive men with urethritis was compared with that in 30 ureaplasma-positive men without urethritis. Results: In men with nonchlamydial NGU and men with Mycoplasma genitalium-negative nonchlamydial NGU, only U. urealyticum subtype 1 (serovars 2, 5, 8, and 9) was detected significantly more often than in men without urethritis. Conclusion: This study suggests that subtype 1 of U. urealyticum (serovars 2, 5, 8, and 9) is associated with NGU independently of Chlamydia trachomatis or M. genitalium.

The last poliomyelitis case associated with a wild poliovirus in Turkey occurred in November 1998. This was the last known case of paralytic poliomyelitis caused by indigenous wild poliovirus in the World Health Organization's European Region. This study investigated the genetic relationships of wild-type 1 polioviruses at the latest period of transmission. A phylogenetic tree was constructed on the basis of the VP1/2A sequence from 14 wild-type 1 polioviruses isolated from Turkey in 1994–1998, along with those from other areas of the world. The Turkey isolates in the latest period of transmission were closely related to each other, forming a cluster distinct from other strains. The results showed that these viruses had been spreading indigenously in the eastern and south-eastern parts of Turkey, and ceased transmission there during 1998. This finding serves as a reference for future poliovirus surveillance both in Turkey and worldwide.

The tiny (T)-strain mycoplasmas, designated in 1974 as Ureaplasma urealyticum, have been divided into 2 species, Ureaplasma parvum (biovar 1) and U. urealyticum (biovar 2), but association of each of these species with nongonococcal urethritis (NGU) remains unclear.The goal of this study was to determine whether U. parvum (biovar 1) or U. urealyticum (biovar 2) is associated with NGU.The prevalences of U. parvum (biovar 1) and U. urealyticum (biovar 2) in 572 patients with urethritis were compared with those in 141 men without urethritis.The prevalence of U. urealyticum (biovar 2) in men with NGU (15.8%) or with nonchlamydial NGU (18.0%) was significantly higher than that in men without urethritis (7.8%). The prevalence of U. parvum (biovar 1) in men with NGU (8.5%) or with nonchlamydial NGU (11.1%) did not differ significantly from that in men without urethritis (13.5%).Our results showed a significant association between U. urealyticum (biovar 2) and NGU. They also suggest that the presence of U. parvum (biovar 1) in the male urethra might be the result of colonization.

Human metapneumovirus (hMPV) has been recently discovered as an etiological agent of acute respiratory infections. Our purpose was to asses the virological and clinical features of children with respiratory infections caused by hMPV. We examined 658 nasopharyngeal swab samples obtained from 637 children with respiratory infections for hMPV by using reverse transcription-PCR (RT-PCR). A total of 268 samples from 637 children were inoculated onto tertiary monkey kidney cells. A total of 36 serum samples (26 in the acute phase and 10 in the convalescent phase) from the 26 hMPV-positive children were tested for immunoglobulin G (IgG) and IgM antibodies to hMPV by using an indirect immunofluorescence assay. We detected hMPV in 57 (8.9%) of the 637 samples by using RT-PCR and isolated 7 (2.6%) hMPV strains of the 268 samples in cell cultures. A total of 12 (46.2%) of 26 hMPV-positive children were suspected to have primary infection with hMPV as determined by an indirect immunofluorescence assay. The infected children were diagnosed as having wheezy bronchitis (36.8%), upper respiratory tract infection (26.3%), bronchitis (22.8%), and pneumonia (14.0%). We showed that two hMPV groups were circulating in different regions during the same period and that reinfection with hMPV frequently occurs in childhood. The RT-PCR test is the most sensitive test for detection of hMPV, and a serological test may be useful to differentiate between primary infection and reinfection with hMPV.

There are several lines of evidence suggesting that specific vaccine therapy with a standard hepatitis B virus (HBV) vaccination reduces HBV replication. The aim of this study was to investigate the anti-viral mechanism of vaccine therapy in chronic hepatitis B patients. Nineteen patients were assigned to receive either vaccine therapy (n = 13) or no treatment as a control (n = 6). Vaccinated patients were analyzed for T cell proliferative responses specific for envelope antigen and cytokine production by antigen-specific T cells. ELISPOT and cytotoxicity assays also were carried out for limited blood samples. Serum HBV DNA levels decreased significantly at 3 months after completion of therapy and thereafter as compared to the baseline ones, and were significantly lower in vaccinated patients than in controls at 12 and 18 months after completion of therapy. Vaccination induced antigen-specific CD4+ T cell proliferative responses in four patients (30.8%). The production of high levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by antigen-specific T cells was found in six patients (46.0%) who showed significantly lower HBV DNA levels in serum at 6 (P = 0.04) and 18 months (P = 0.005) after completion of therapy than those without high levels of cytokine production. Vaccination did not induce antigen-specific CD8+ T cells or cytotoxic T cells. These results suggest that envelope-specific CD4+ T cells may control directly HBV replication by producing anti-viral cytokines rather than providing help for cytotoxic T cells in therapeutic vaccination against chronic HBV infection. J. Med. Virol. 71:376–384, 2003. © 2003 Wiley-Liss, Inc.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Objective Hepatitis C virus (HCV) E2 protein was recently reported to have a double-stranded RNA-activated protein kinase–eukaryotic initiation factor 2α (PKR-eIF2α) phosphorylation homology domain (PePHD); PKR is induced by interferon (IFN). PePHD interacts with PKR and inactivates it. PePHD could be a predictor for IFN response, like the interferon sensitivity determination region (ISDR) of HCV NS5A. Several groups reported that PePHD is conserved, and mutations in this region do not correlate with IFN response. In this study, we further investigated the amino acid variation of PePHD among four major genotypes and its correlation with IFN response. Methods We enrolled 74 patients for this study and determined PePHD sequence of HCV derived from sera of patients infected with HCV genotype 1a (1 patient; nonresponder [NR]), 1b (36 patients; 4 complete responders [CR], 32 NR), 2a (29 patients; 17 CR, 12 NR), and 2b (8 patients; 3 CR, 5 NR). We also analyzed mutations in ISDR of HCV genotype 1b in 31 patients. Results PePHD had several variations among four genotypes investigated. In patients infected with HCV genotype 1b, PePHD sequence was well conserved and seemed to have no correlation with IFN response. Mutations in ISDR were correlated with IFN response. In patients with HCV genotypes 2a and 2b, PePHD had multiple variations, and one particular motif, “RGQQ-” at the N-terminus, showed a close correlation with IFN resistance. All eight patients with HCV containing this motif were IFN nonresponders. Conclusions IFN resistance of HCV correlates with its “RGQQ-” motif at the N-terminus of PePHD in HCV genotype 2a and 2b. PePHD of HCV could be a predictor of IFN resistance in patients infected with HCV genotype 2a and 2b.

We present a method for detecting the presence of Mycoplasma genitalium , Mycoplasma hominis , Ureaplasma parvum , and Ureaplasma urealyticum organisms, which are thought to be associated with nongonococcal urethritis (NGU) and other genitourinary infections, in clinical samples. This method consists of PCR amplification of a part of the 16S rRNA gene followed by 96-well microtiter plate hybridization assay using four species-specific capture probes to detect the targets. To test the efficacy of this method, we applied it to the detection of the four species in the urine of patients with NGU. There were no cross-reactions with other human mycoplasmas or ureaplasmas, and the PCR-microtiter plate hybridization assay detected as few as 10 copies of the 16S rRNA gene of each of the four species. Based on these results, this PCR-microtiter plate hybridization assay can be considered an effective tool for the diagnosis of genitourinary infections with mycoplasmas or ureaplasmas.

To the Editor: Mycoplasma genitalium is now recognized as a serious pathogen in sexually transmitted infections (1,2). Azithromycin regimens have been commonly used for treatment of M. genitalium infections (3). However, failure of azithromycin treatment has been reported in cases of M. genitalium–positive nongonococcal urethritis (NGU) (4,5), and macrolide-resistant strains of M. genitalium have been isolated from case-patients in Australia, Sweden, and Norway for whom azithromycin treatment has failed (4,5). In these strains, mutations in the 23S rRNA gene were associated with macrolide resistance, and mutations in ribosomal protein genes L4 and L22 were also found (5). Surveillance for antimicrobial resistance of M. genitalium is essential to identify antimicrobial resistant strains and to then determine appropriate treatment. Coculture of patient specimens with Vero cells has improved the primary isolation rate of M. genitalium from clinical specimens and offered some current clinical strains for antimicrobial drug susceptibility testing (6). To determine their antimicrobial susceptibilities, a molecular real-time PCR method has been developed (7,8). However, isolating M. genitalium from clinical specimens and antimicrobial drug susceptibility testing of clinical isolates remain labor-intensive, time-consuming tasks. In addition, no methods are available to directly determine antimicrobial drug susceptibilities of M. genitalium in clinical specimens. To monitor macrolide susceptibilities in clinical strains of M. genitalium in Japan, therefore, we examined M. genitalium DNA found in the urine of men with NGU for the presence of macrolide resistance–associated mutations in the 23S rRNA gene and the ribosomal protein genes L4 and L22. This retrospective study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan. We collected pretreatment urine specimens from 308 men with NGU who had visited a urologic clinic (iClinic) in Sendai, Japan, during 2006 through 2008 and stored the specimens at –70°C. Each man gave informed consent. Twenty-five of 58 urine specimens confirmed to be positive for M. genitalium by PCR-based assay were randomly chosen for this study and subjected to DNA purification. The 23S rRNA gene and the ribosomal proteins genes L4 and L22 of M. genitalium were amplified from the purified DNA by PCR as reported previously and then sequenced (5). In 1 specimen, we found an A-to-G transition at nucleotide position 2072 in the 23S rRNA gene of M. genitalium, corresponding to position 2059 in Escherichia coli (Table). An A2059 (E. coli numbering) residue in region V of the 23S rRNA gene is critical for the binding of macrolides (9). Mutations of A2058, A2059, and other 23S rRNA residues within the macrolide-binding site can confer a high-level resistance to macrolides in several bacterial species, including M. genitalium (5,9). Therefore, M. genitalium strains that harbor the A2059G (E. coli numbering) mutation in the 23S rRNA gene could be highly macrolide resistant. We also found a T-to-G transition at nucleotide position 2199 in the 23S rRNA gene of M. genitalium, corresponding to position 2185 in E. coli, in 3 specimens, but this mutation has not been associated with macrolide resistance in other bacterial species (9). Table Mutations in the 23S rRNA gene and amino acid changes in L4 and L22 ribosomal proteins of 25 Mycoplasma genitalium strains in the pretreatment urine specimens of men with nongonococcal urethritis, Japan We found amino acid changes in L4 and L22 ribosomal proteins in M. genitalium in 9 specimens. L4 and L22 ribosomal proteins each have extended loops, which converge to form a narrowing in the exit tunnel adjacent to the macrolide-binding site (10). Therefore, macrolide resistance–associated missense mutations in L4 and L22 tend to be localized to Gln62–Gly66 in L4 and Arg88-Ala93 in L22 of E. coli, which are closest to the macrolide-binding site (10). All of the amino acid changes in L4 of M. genitalium found in this study corresponded to those at the downstream regions from Gln62-Gly66 in L4 of E. coli. Of the amino acid changes in L22 of M. genitalium, the only Gly93Glu change found in M. genitalium harboring the A2059G (E. coli numbering) mutation in the 23S rRNA gene was located within the region corresponding to Arg88-Ala93 in L22 of E. coli. In this strain, therefore, the Gly93Glu change in L22 might contribute to the increase of macrolide resistance. The patient with NGU, whose specimen exhibited this strain of M. genitalium that harbored both the A2059G (E. coli numbering) mutation in the 23S rRNA gene and in which the Gly93Glu change in L22 was detected, was given a single dose of 1 g azithromycin and was clinically cured of NGU. However, the present study suggests that M. genitalium strains with high-level macrolide resistance might have already emerged in clinical settings in Japan. The emergence and spread of such a clinical mutant could threaten the ability of macrolides to treat M. genitalium infections. We should continue monitoring macrolide resistance of M. genitalium clinical strains. The nonculture approach used in our study will be useful until culturing of mycoplasmas from clinical specimens and antimicrobial drug susceptibility testing can be performed easily in laboratories.

Epstein–Barr virus (EBV) has been shown to infect T lymphocytes and to be associated with a chronic active infection (CAEBV), which has been recognized as a mainly non-neoplastic T-cell lymphoproliferative disorder (T-cell LPD). The systemic distribution of EBV genomes was studied, by real-time PCR, in multiple tissues from six patients with CAEBV, including three patients with T-cell LPD, one patient with B-cell LPD and two patients with undetermined cell-type LPD. There were extremely high loads of EBV genomes in all tissues from the patients. This reflects an abundance of circulating and infiltrating EBV-infected cells and a wide variety of clinical symptoms in the affected tissues. We chose one sample from each patient that was shown by real-time PCR to contain a high load of EBV genomes and examined the expression of EBV latent genes by RT–PCR. EBER1 and EBNA1 transcripts were detected in all samples. Only one sample also expressed EBNA2, LMP1 and LMP2A transcripts in addition to EBER1 and EBNA1 transcripts. Two of the remaining five samples expressed LMP1 and LMP2A transcripts. One sample expressed LMP2A but not LMP1 and EBNA2 transcripts. Another sample expressed EBNA2 but not LMP1 and LMP2A transcripts. The other sample did not express transcripts of any of the other EBNAs or LMPs. None of the samples expressed the viral immediate-early gene BZLF1. These results showed that EBV latent gene expression in CAEBV is heterogeneous and that restricted forms of EBV latency might play a pathogenic role in the development of CAEBV.

Epidemics of acute hemorrhagic conjunctivitis (AHC) caused by a variant of coxsackievirus A24 (CA24v) reappeared in Taiwan in 1990 and 1994, following the first two epidemics of 1985--86 and 1988--89. To analyze the genetic diversity of recent CA24v in Taiwan, 7 Taiwanese strains isolated during the 1990--94 period were studied together with one Japanese and two Thai strains isolated in 1993. A fragment of 674 nucleotides between the carboxy terminal 3A and the amino terminal 3D polymerase, including the entire 3C protease (3C(pro)), was amplified by a reverse transcription-polymerase chain reaction (RT-PCR) and the nucleotide sequences were determined. In the 549 nucleotides (183 amino acids) of the entire 3C(pro), we found nucleotide differences at 80 positions between 10 strains and the prototype strain, EH24/70, one of the earliest strains of CA24v. Most of the nucleotide changes were synonymous substitutions and only nine amino acid changes were found. The nucleotide sequence homologies among 71 strains worldwide were 88-100%. These 71 nucleotide sequences were then analyzed by Neighbor-joining method and phylogenetically separated into three distinct genotypes. Genotype I consisted of early strains isolated in 1970--71 from Singapore and Hong Kong. Genotype II included isolates from Singapore and Thailand obtained in 1975. Genotype III comprised strains from the eastern hemisphere isolated in 1985--94 from Japan, Taiwan, China, Hong Kong, Thailand, Singapore, Pakistan and Ghana. They were further divided chronologically into six clusters. The recent isolates from Taiwan obtained in 1985/1986, 1988/1989 and 1990--94 were classified into genotype III Clusters 1, 5, and 6 respectively. The evolutionary rate was re-estimated to be 3 x 10(- 3) 30 years after the emergence of the virus.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 109 RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r = 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections. J. Med. Virol. 63:29–34, 2001. © 2001 Wiley-Liss, Inc.

Enterovirus 71 has been associated with several outbreaks, as well as sporadic cases, of central nervous system infection and has a worldwide distribution. Seven children with encephalitis and five with aseptic meningitis caused by Enterovirus 71 were seen at Otsu Municipal Hospital during the summer of 1997. The infections were confirmed serologically, although detection of the viral genome in cerebrospinal fluid was unsuccessful. Seven children were diagnosed as having hand-foot-and-mouth syndrome, two were diagnosed as having herpangina, and three patients younger than 12 months old developed no eruptions. The skin or mucosal manifestations of this outbreak demonstrated considerable variation. The Enterovirus 71 strain that caused the outbreak had a strong neurovirulent tendency. Among the patients with encephalitis, symptoms originating from the impairment of diencephalon were seen in four patients, and those originating from cerebellar impairment were seen in two patients. Brain magnetic resonance imaging in one patient revealed an abnormality in the pons. The neurologic manifestations associated with Enterovirus 71 infection may be characterized by involvement of the cerebellum, brainstem, and diencephalon. Enterovirus 71 is one of the pathogenic viruses that cause hand-foot-and-mouth syndrome, as well as a variety of other clinical manifestations. The most important of these is neurologic disease, especially in infants and young children.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Purpose To investigate the serotypes of adenovirus causing conjunctivitis in Hanoi, Vietnam. Design Clinical diagnosis of adenoviral conjunctivitis and laboratory-based experimental study. Methods We collected 21 conjunctival swabs from 21 different patients with a clinical presentation compatible with adenoviral conjunctivitis, in Hanoi, Vietnam. Immunochromatography and real-time polymerase chain reaction (PCR) were performed to detect human adenovirus (HAdV). The sequence of PCR products was analyzed to determine the serotype of HAdV. Results Of 21 samples, HAdV DNA was detected in 14 samples (66.7%) by real-time PCR. The serotype analysis showed HAdV-8 in 11 samples (78.6%), HAdV-3 in two samples (14.3%), and HAdV-37 in one sample (7.1%). Of 11 HAdV-8 samples, one sample (9.1%) was prototype, and the other 10 samples (90.9%) had identical nucleotide sequence and were identified as a variant of HAdV-8. Conclusions HAdV-8 was found to be the predominant serotype in Hanoi, Vietnam. Most of the HAdV-8 samples were a variant of HAdV-8.

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are known to be major causative agents of hand-foot-and-mouth disease prevalent in summer in Japan. Discrimination and identification of these viruses were often hampered by a nonneutralizable or nontypable virus. Therefore, a Southern blot hybridization that utilizes mixed probes specific to serotype was developed. Firstly, an approximately 650 bases spanning 5'-noncoding region to one third of VP2 including entire VP4 was amplified with a set of primers containing enterovirus common sequences and a genomic RNA as template. Secondary, the nucleotide sequences were determined using seven CA16 and eighteen EV71 strains including the standard strains, and the deduced amino acid sequences of VP4 were searched to find residues which are conserved in the same serotypes but diverged among different serotypes. Candidate positions for the mixed probes were defined at the carboxyl terminus of VP4. Thirdly, Southern blot analyses were carried out using thirty-nine enterovirus standard strains, seven CA16 isolates and sixty-six EV71 isolates previously identified by the neutralization test. The results revealed that each mixed probe exclusively bound to the homologous DNAs but not to the heterologous ones. In an attempt to determine serotypes without virus isolation, clinical specimens from hand-foot-and-mouth disease were examined. Of 78 throat swabs and 15 vesicular fluids, 71 (91.0%) and 13 (86.7%) specimens were clearly identified, indicating that the method described here offer advantages over the traditional neutralization assay: It is rapid, specific and less labor-consuming.

Hepatitis C Virus (HCV) is the major cause of parentally transmitted non-A, non-B hepatitis. We studied the incidence of HCV Viremia in blood donors, hemophiliacs and patients with chronic liver disease who are positive for antibodies to HCV, and then correlated the HCV genotypes among the three groups. 23 blood donors, 10 hemophiliacs and 97 patients with chronic liver disease were found to be positive for anti-HCV during this study period from June 1993 to December 1993. Only 3 (13%) blood donors, 6 (60%) hemophiliacs and 71 (73%) patients with chronic liver disease were found to be viremic when tested for HCV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The low incidence of viremia among blood donors may be due to any one of the following three reasons. 1, the level of viremia was below the level of detection. 2, the viremia was intermittent with persistent infection. 3, the majority of cases represented resolved infection. The HCV genotypes were heterogeneous among the three groups. All the blood donors with viremia and 35 (50%) of patients with chronic liver disease, belonged to type II (1b). However only one (17%) of the hemophiliacs belonged to type II (1b). Studies have shown that the genotype I(1a) is the predominant type in the USA and Europe, whereas type II(1b) is more frequent in the Far East. It is also suggested that type II (1b) is associated with non-responsiveness to interferon therapy. Our hemophiliacs were treated with imported coagulation factors, thus they were probably exposed to the genotypes in the west. There was significant difference in the incidence of HCV type II (1b) among local blood donors and hemophiliacs (P = 0.005). However the difference between the hemophiliacs and the patients with chronic liver disease was not statistically significant. The number of patients in this study was too small to draw any firm conclusions. However the findings highlight the importance of studying the genotypes of patients with Hepatitis C infection due to their relevance in the management of these cases with interferon therapy.

We performed two courses of interferon-beta (IFN-beta) to a child with chronic hepatitis C. A complete response was not obtained by the first interferon treatment, however, the results of the second treatment differed from those of the first. Hepatitis C virus (HCV)-RNA remained negative and both aspartate aminotransferase and alanine aminotransferase levels remained normal after completion of the second course. From these results we estimated that HCV-RNA levels before IFN therapy could be significantly associated with the efficacy of this treatment. The serum level of HCV-RNA was 10(6) copies/50 microL before the first treatment, but was 10(3) copies/50 microL before the second course. We conclude that IFN therapy to children with hepatitis C should always be directed at providing a cure. Even if the clinical effects of the first course are minimal decreasing quantities of HCV-RNA still offer hope for cure by subsequent readministration.

We report a familial clustering case of hepatitis delta. All of the members of this family had evidence of past infection of hepatitis B. We investigated the hepatitis delta, three of the members had positive serological hepatitis delta markers. We assayed by polymerase chain reaction the primers corresponding to hepatitis delta antigen. The results of polymerase chain reaction was three positive. The 2nd polymerase chain reaction was used, two geno-type specific primers one was for Japanese S type the other was for Japanese M type7). Three were positive the 2nd polymerase chain reaction for Japanese M, one was negative for all of hepatitis delta polymerase chain reaction.

Summary Hepatitis E virus (HEV), a causative agent of human hepatitis E, is transmitted through an oral-fecal route, often by contaminated drinking water. Serum specimens were collected from 112 non-A, -B, and –C acute hepatitis patients from 1989 to 2004 in Japan. Of these, 24 patients were found to be positive for anti-HEV IgM and diagnosed with acute Hepatitis E. Seventeen of these patients had a clear history of traveling abroad before disease onset and were counted as cases of imported HEV infection. HEV RNA was detected in 16 of these imported cases, and the nucleotide sequences similar to those of HEV detected in India, Bangladesh, and China were identified. By phylogenetic analysis, the remaining imported case appeared to have been imported from India, even though the patient's travel history was uncertain. These results indicated that some sporadic cases of hepatitis E in Japan are caused by imported HEV, and that phylogenetic analyses enable us to identify the country or area where a patient has been infected.

Objective A single 1 g dose regimen of azithromycin has been recommended for the treatment of Mycoplasma genitalium infections. The authors evaluated whether this regimen could select M genitalium strains with macrolide resistance after treatment for M genitalium -positive non-gonococcal urethritis. Methods In seven men with non-gonococcal urethritis, who were infected with M genitalium without macrolide resistance-associated mutations but experienced microbiological azithromycin treatment failure, M genitalium DNAs in their post-treatment urine specimens were examined for mutations in the 23S rRNA gene and the ribosomal protein genes of L4 and L22. To assess the relatedness of M genitalium strains before and after treatment, their DNAs in pretreatment and post-treatment urine were genotyped by analysing short tandem repeats of an AGT/AAT unit in the MG309 gene and single nucleotide polymorphisms in the MG191 gene. Results In four of seven patients, M genitalium in post-treatment urine had an A-to-G transition at nucleotide position 2071 or 2072, corresponding to 2058 or 2059 in the 23S rRNA gene of Escherichia coli . In one of the four strains, Pro81Ser in the ribosomal protein L4 accompanied the mutation in the 23S rRNA gene. The genotyping of M genitalium DNAs suggested that these four post-treatment strains were selected from the respective closely related or identical pretreatment strains without macrolide resistance-associated mutations by the treatment. Conclusions The single 1 g dose treatment of azithromycin could select M genitalium strains harbouring macrolide resistance-associated mutations. For M genitalium , this regimen might increase the risk of macrolide resistance selection after treatment.

Nucleotide substitutions in the viral-encoded proteinase 3C (3Cpro) region (549 nucleotides) of the RNA genome of a coxsackievirus A24 variant (CA24v), one of the agents causing acute hemorrhagic conjunctivitis (AHC), were studied using 32 isolates collected from the Eastern hemisphere in 1970-1989. Based on regression analysis of nucleotide differences among isolates, the nucleotide substitution rate of CA24v 3Cpro was estimated to be 3.7 x 10(-3)/nucleotide/year. A phylogenetic tree constructed by the modified unweighted pair group method using arithmetic averages (UPGMA) indicated that CA24v had evolved from a common ancestor which appeared in one focal place in November 1963 +/- 21 months, about 7 years before the first isolation of CA24v in Singapore. The tree also revealed that all the recent epidemic isolates in 1985-1989 including Asian and Ghanian strains diverged from each other after 1981. This finding is consistent with the evidence that AHC due to CA24v had been confined to Southeast Asia and the Indian subcontinent until 1985, then suddenly and explosively spread to other areas where no CA24v isolations had been reported.

We have reported a novel human adenovirus (HAdV) that has caused a nationwide epidemic of keratoconjunctivitis (EKC) in Japan ([11][1]). This virus has been characterized serologically and genetically as a novel serotype, and we propose naming it HAdV-54. The GenBank accession number of the

Human metapneumovirus (hMPV) strains are classified into two genetic groups, A and B, each of which is further divided in two genetic subgroups, A1, A2, B1 and B2. hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. Although the amino acid sequences of hMPV F protein are highly conserved, those of the G protein are highly variable with low amino acid identity between the two groups. To address the antigenic variation between the genetic subgroups, we developed an immunofluorescence assay (IFA) method using Trichoplusia ni (Tn5) insect cells infected with each recombinant baculovirus-expressed hMPV G (Bac-G) protein of the four genetic subgroups. The titre of each antibody to the four Bac-G proteins was measured by the IFA in 12 paired serum samples obtained from children infected with hMPV of each genetic subgroup. Although 11 of the 12 acute-phase serum samples in paired samples were negative for the antibody to any Bac-G proteins, all of the convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Since the antibody response to hMPV G protein was transient and genetic subgroup-specific without cross-reactivity, four genetic subgroups on the basis of hMPV G protein could be identified as different serotypes. This assay may be useful for the study of immune responses of humans to different hMPV strains, especially for clarifying the risk of reinfection with hMPV.

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.

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.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) are thought to be effective in limiting viral spread and in clearing virus during infection. Therefore, we attempted to establish HCV-specific CTL and identify novel HCV-specific CTL epitopes in a patient with acute hepatitis C by a novel screening method using recombinant vaccinia viruses (rVV) and synthetic peptides. CD8(+)CD45RA(-) T cells (memory T cells) were isolated from peripheral blood mononuclear cells (PBMC) of a patient with acute hepatitis C. HCV-specific CTL were cloned at limited dilutions and tested for HCV-specific CTL activity using a standard (51)Cr release assay. CTL assay was performed using rVV expressing regions of HCV-J, and overlapping and truncated synthetic peptides from HCV-J. CTL recognizing the NS3 region were isolated by (51)Cr release assay with rVV-HCV. Isolated CTL were restricted by HLA class I molecules B(*)5603. We confirmed that isolated CTL recognized 8-mer amino acids in the NS3 region of HCV-J by (51)Cr release assay with overlapping and truncated synthetic peptides. In conclusion, we isolated HCV-specific CTL restricted by HLA-B(*)5603 and identified a novel HCV-specific CTL epitope (IPFYGKAI, amino acids 1373-1380) in the NS3 region. The identified HCV-specific CTL epitope might be useful for HCV therapy.

Human bocavirus (HBoV), a newly cloned human virus of the genus Bocavirus , was detected by PCR from nasopharyngeal swab samples (8 of 318; 5.7%) collected from children with lower respiratory tract infections. HBoV may be one of the causative agents of lower respiratory tract infections in young children.

More than 20 years ago, Mycoplasma genitalium was first isolated from men with nongonococcal urethritis (NGU). Some strains of Mycoplasma and Ureaplasma are associated with NGU or other genitourinary infections. It is reasonable to suggest that mycoplasmas and ureaplasmas may increase the development of acquired immunodeficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected patients. This study investigated a rapid and reliable diagnostic method, based on a LightCycler SYBER green I real-time PCR assay and phylogenetic analysis, for detecting mycoplasmas and ureaplasmas in urine from HIV-1-infected patients. Primers were designed to amplify a 711-bp fragment of the 16S rRNA gene. Positive PCR-amplified products were further sequenced, and a phylogenetic tree was constructed by neighbor joining of the V4 and V5 sequences from 15 prototypes strains. PCR-positive samples were detected in 17 (9.1%) urine samples, their prototypes being as follows: 2/17 (11.2%) M. genitalium, 1/17 (5.9%) M. hominis, 12/17 (70.6%) Ureaplasma urealyticum, 1/17 (5.9%) mixed M. genitalium and M. hominis, and 1/17 (5.9%) M. hominis and U. urealyticum, respectively. Mycoplasma and Ureaplasma load for each specimen was 3.8 x 10 copies/ml to 7.2 x 10(3) copies/ml. Overall, this study demonstrates that real-time PCR and phylogenetic analysis are the fastest available methods for the detection of mycoplasmas in the urogenital tract.

The aim of this study was to assess the tolerability and efficacy of N-chlorotaurine (NCT), an endogenous antimicrobial agent, in epidemic keratoconjunctivitis. In a prospective double-blind, randomized phase 2b study, the infected eyes were treated for 7 days with eye drops containing 1% aqueous solution of N-chlorotaurine (33 subjects) or gentamicin (27 subjects, control group). Adenovirus types 3, 4, 8, 19, and 37 were detected in 39 subjects (65%), enteroviruses in 8 (13.3%), and staphylococci in 5 (8.3%). Subjective and objective symptoms were scaled and added to a subjective and objective score, respectively, on day 1 (baseline), day 4, and day 8. Analyzing the whole study population, the subjective score on day 8 was lower in the NCT group (P = 0.016), whereas there were no differences in the objective score. However, in severe infections caused by adenovirus type 8 (n = 20) both the subjective and objective score were lower in the NCT group on day 4 (P = 0.003 and 0.015, respectively), which was also true for the subjective score on day 8 (P = 0.004) in this subgroup. The frequency of subepithelial infiltrates was similar in both groups. N-chlorotaurine was well-tolerated, shortened the duration of illness, and seems to be a useful causative therapeutic approach in severe epidemic keratoconjunctivitis.

Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.

Aim: To use polymerase chain reaction (PCR)-microtiter plate hybridization assays to detect Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum (biovar 1) and Ureaplasma urealyticum (biovar 2) in first-voided urine specimens from patients with non-gonococcal urethritis (NGU). Methods: A total of 153 male patients with NGU, who visited one of 24 clinics in Japan, were recruited for this study. All were examined using PCR-microtiter plate hybridization assays for the presence of M. genitalium, M. hominis, U. parvum (biovar 1) and U. urealyticum (biovar 2) in first-voided urine specimens. They were also examined for the presence of Chlamydia trachomatis. Results: Of these 153 patients, 73 (47.7%) were positive for C. trachomatis. Overall, the prevalence was 17.0% for M. genitalium, 16.3% for U. urealyticum (biovar 2), 7.8% for U. parvum (biovar 1) and 2.6% for M. hominis. In the 80 patients with non-chlamydial NGU, the prevalence of M. genitalium, U. urealyticum (biovar 2), U. parvum (biovar 1) and M. hominis was 23.8%, 18.8%, 8.8% and 2.6%, respectively. Conclusions: This study shows the prevalence of mycoplasmas and ureaplasmas in NGU in Japan. M. genitalium and U. urealyticum (biovar 2) might be pathogens of NGU and could be associated with persistent and recurrent urethritis. When patients with NGU are treated, such pathogens should be taken into account. This PCR-microtiter plate hybridization assay provides a useful method for diagnosing NGU caused by M. genitalium and U. urealyticum (biovar 2).