Tsuguto Fujimoto

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

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

Evaluation of a Bedside Immunochromatographic Test for Detection of Adenovirus in Respiratory Samples, by Comparison to Virus Isolation, PCR, and Real-Time PCR

ABSTRACT An immunochromatography (IC) kit for human adenovirus (HAdV) was evaluated with 138 patient nasopharyngeal samples. The samples were collected at a sentinel clinic in Japan from January through June 2003. Patients were diagnosed by clinical manifestation of pharyngoconjunctival fever (n = 38) or exudative tonsillitis (n = 100). The IC kit was positive for 84% (116 of 138) of patients diagnosed at bedside. The remaining extract solution of the IC kit test was transferred into maintenance medium and tested via laboratory diagnoses. The IC kit had 95% sensitivity (116 of 122 patients) with HAdV isolation (isolation) as the standard and 91% sensitivity (116 of 128 patients) with PCR as the standard. All of the IC kit-positive samples were isolation and PCR positive. Similarly, all the isolation-positive samples were PCR positive. Twenty-two IC kit-negative samples were evaluated by real-time PCR. Six samples were IC kit negative and isolation positive and contained 3.8 × 107 to 2.5 × 109 copies of the HAdV genome/ml. Five samples that were only PCR positive contained 3.0 × 104 to 3.8 × 105 copies of the HAdV genome/ml, but one sample was real-time PCR negative. We conclude that the IC kit is a useful bedside diagnostic tool for HAdV infections because it has 95% sensitivity (compared to isolation), but a negative result does not always rule out HAdV infection.

Novel Human Adenovirus Strain, Bangladesh

We report a novel human adenovirus D (HAdV-65) isolated from feces of 4 children in Bangladesh who had acute gastroenteritis. Corresponding genes of HAdV-65 were related to a hexon gene of HAdV-10, penton base genes of HAdV-37 and HAdV-58, and a fiber gene of HAdV-9. This novel virus may be a serious threat to public health.

Complete Genome Analysis of a Novel Intertypic Recombinant Human Adenovirus Causing Epidemic Keratoconjunctivitis in Japan

ABSTRACT For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC.

Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence.

Human adenovirus type 53 (HAdV-53) has commonly been detected in samples from epidemic keratoconjunctivitis (EKC) patients in Japan since 1996. HAdV-53 is an intermediate virus, containing hexon-chimeric, penton base and fiber structures similar to HAdV-22 and -37, HAdV-37 and HAdV-8, respectively. HAdV-53-like intermediate strains were first isolated from EKC samples in Japan in the 1980s. Here, the complete genome sequences of three such HAdV-53-like intermediate strains (870006C, 880249C and 890357C) and four HAdV-53 strains were determined, and their relationships were analysed. The seven HAdV strains were classified into three groups, 870006C/880249C, 890357C and the four HAdV-53 strains, on the basis of phylogenetic analyses of the partial and complete genome sequences. HAdV strains within the same group showed the highest nucleotide identities (99.87-100.00 %). Like HAdV-53, the hexon loop 1 and 2 regions of 870006C, 880249C and 890357C showed the highest identity with HAdV-22. However, these strains did not show a hexon-chimeric structure similar to HAdV-22 and -37, or a penton base similar to HAdV-37. The fiber genes of 870006C and 880249C were identical to that of HAdV-37, but not HAdV-8. Thus, the three intermediate HAdVs isolated in the 1980s were similar to each other but not to HAdV-53. The recombination breakpoints were inferred by the Recombination Detection Program (rdp) using whole-genome sequences of these seven HAdV and of 12 HAdV-D strains from GenBank. HAdV-53 may have evolved from intermediate HAdVs circulating in the 1980s, and from HAdV-8, -22 and -37, by recombination of sections cut at the putative breakpoints.

Genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis.

ABSTRACT A novel virus of the species human adenovirus D, HAdV-67 (P-New/H9/F25), was first isolated from diarrheal feces of six children in Dhaka City, Bangladesh. The genome of this novel virus may be composed of multiple recombinations among HAdV-9, HAdV-25, HAdV-26, HAdV-33, HAdV-46, and an unknown human adenovirus D which was an origin of HAdV-67.

Surveillance of Adenovirus D in patients with epidemic keratoconjunctivitis from Fukui Prefecture, Japan, 1995–2010

Human adenoviruses species D (HAdV‐D) are known to cause severe epidemic keratoconjunctivitis. However, the isolation rate of HAdV‐D is not high, because HAdV‐D is usually slow to propagate. Although new types of HAdV‐D have been reported, accurate surveillance has not been performed because of difficulties in culturing the viruses and lack of a practical identification method. In this study, HAdV‐Ds were detected and identified from patients with epidemic keratoconjunctivitis in the Fukui Prefecture during 1995–2010 by PCR, loop‐mediated isothermal amplification (LAMP) of DNA, and conventional virus isolation and neutralization tests. All samples were subjected to culture and PCR and LAMP. A total of 124 strains of HAdV‐D were detected from 157 patients with epidemic keratoconjunctivitis. The strains consisted of the following types: D8 (n = 8), D19 (n = 4), D37 (n = 40), D53 (n = 5), D54 (n = 66), and D56 (n = 1). Among these, D53, D54, and D56 are new types that have been reported recently. The results of this study demonstrated that new types of HAdV‐D caused epidemic keratoconjunctivitis during 1995–2010, and included an outbreak of keratoconjunctivitis caused by HAdV‐D54. The LAMP method was able to detect and identify HAdV‐D53 and HAdV–D54 in 1 hr, and may therefore be applicable for use at the bedside. J. Med. Virol. 84:81–86, 2011.

Molecular Epidemiology of Echoviruses 11 and 13, Based on an Environmental Surveillance Conducted in Toyama Prefecture, 2002-2003

ABSTRACT Nineteen echovirus 11 (E11) and 12 E13 isolates were isolated from three rivers in Toyama Prefecture, Japan, during an environmental surveillance conducted from April 2002 to March 2003. The nucleotide sequences of E13 isolates were closely related to those from patients with aseptic meningitis, with less than 1.3% divergence in the VP1 region of the viral capsid gene, and belonged to the same clade responsible for a worldwide outbreak that started in 2000. In contrast, E11 isolates were clustered into three genomic groups and were not closely related to echovirus strains isolated from patients. These results suggest that the combination of both virus isolation from environmental sources and phylogenetic analysis could be complementary assessment approaches to trace prevalent and minor circulating enteroviruses in the human population.

Single-Tube Multiplex PCR for Rapid and Sensitive Diagnosis of Subgenus B and Other Subgenera Adenoviruses in Clinical Samples

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non‐nested polymerase chain reaction (PCR). Sequences of the conserved hexon‐coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single‐tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non‐Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty‐seven out of 71 (94%) Sub B Ad culture‐positive samples, and 15 out of 19 (79%) Sub C or E‐positive samples amplified products of the expected size. Two of 20 (10%) culture‐negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non‐Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.

Human adenovirus type 8 genome typing.

Human adenovirus type 8 (HAdV-8) is a major causative agent of epidemic keratoconjunctivitis, which is frequently associated with community, industrial and nosocomial outbreaks. Restriction endonuclease (RE) analysis discriminates HAdV-8 isolates into genome types, making it possible to correlate between genomic variants, virulence and outbreak potential. RE analysis is performed using two sets of classification criteria, an Asian and a European system. So far, genome types HAdV-8A-8K and HAdV-8/D1-D12 have been included in the Asian and European classifications, respectively. Conventionally followed RE analysis has some inherent problems, such as the use of a neutralization test for HAdV-8 typing, which may misidentify some recombinant adenoviruses as HAdV-8 due to cross-reaction, the lack of a complete restriction profile for all genome types for purposes of comparison, and the absence of enzyme codes in the Asian classification system. In this review, we propose typing of HAdV-8 with phylogenetic analysis of the hexon and fibre genes prior to RE analysis due to the emergence of many recombinant types. Schematic restriction profiles for both classification systems were created by compiling all the published reports on genome types, and enzyme codes were included for the Asian classification system. The updated and simplified stepwise approach for HAdV-8 genome typing presented here could be useful for identifying either existing genome types or novel ones.