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

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.

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.

High frequency of amantadine-resistant influenza A (H3N2) viruses in the 2005-2006 season and rapid detection of amantadine-resistant influenza A (H3N2) viruses by MAMA-PCR.

Using the newly designed mismatch amplification mutation assay (MAMA) PCR, we demonstrated the high frequency of amantadine-resistant influenza A (H3N2) viruses isolated during the 2005-2006 season by detecting the mutation at amino acid position 31 of the M2 protein (S31N). Further, phylogenetic analyses of the HA1 sequences of the S31N viruses revealed that they comprised a clonal lineage that would result in the common characteristic amino acid changes at positions 193 (Ser to Phe) and 225 (Asp to Asn) of the HA protein. We also demonstrated that the S31N/S193F/D225N viruses had already emerged in Aichi Prefecture by the end of the previous 2004-2005 season.

Genetic and antigenic diversity among noroviruses.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Ability of Shiga toxin-producing Escherichia coli and Salmonella spp. to survive in a desiccation model system and in dry foods.

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35 degrees C for 24 h in paper disks. At an inoculum level of 10(7) CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10(3) to 10(4) CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10(2) CFU/disk). After 22 to 24 months of subsequent storage at 4 degrees C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10(3) to 10(4) CFU/disk). In contrast to the case for storage at 4 degrees C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25 degrees C and 35 degrees C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70 degrees C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25 degrees C.

Close correlation of streptococcal DNase B (sdaB) alleles with emm genotypes in Streptococcus pyogenes.

DNase B is a major nuclease and a possible virulence factor in Streptococcus pyogenes. The allelic diversity of streptococcal DNase B (sdaB) gene was investigated in 83 strains with 14 emm genotypes. Of the 15 alleles identified, 11 alleles carried only synonymous nucleotide substitutions. On the other hand, 4 alleles had a non-synonymous substitution other than synonymous substitutions, resulting in the substitution of a single amino acid. The distribution of each allele was generally emm genotype-specific. Only sdaB7 was found in both emm2 and emm4. The promoter region was highly conserved and DNase B protein was similarly expressed in all alleles.

Expression and antigenicity of virus-like particles of norovirus and their application for detection of noroviruses in stool samples.

Human noroviruses (NoVs), members of the genus Norovirus in the family Caliciviridae, are the leading agents of nonbacterial acute gastroenteritis worldwide. Human NoVs are currently divided into at least two genogroups, genogroup I (GI) and genogroup II (GII), each of which contains at least 14 and 17 genotypes. To explore the genetic and antigenic relationship among NoVs, we expressed the capsid protein of four genetically distinct NoVs, the GI/3 Kashiwa645 virus, the GII/3 Sanbu809 virus, the GII/5 Ichikawa754 virus, and the GII/7 Osaka10-25 virus in baculovirus expression system. An antigen enzyme-linked immunosorbent assay (ELISA) with hyperimmune serum against the four recombinant capsid proteins and characterized previously three capsid proteins derived from GI/1, GI/4, and GII/12 was developed to detect the NoVs antigen in stools. The antigen ELISA was highly specific to the homotypic strains, allowing assignment of a strain to a Norovirus genetic cluster within a genogroup.

Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b.

A novel gene for quinolone resistance was cloned from a transferable plasmid carried by a clinical isolate of Shigella flexneri 2b that was resistant to fluoroquinolones. The plasmid conferred low-level resistance to quinolones on Escherichia coli HB101. The protein encoded by the gene showed 59% amino acid identity with Qnr.

Molecular mechanisms of high level tetracycline-resistance in group A streptococcal isolates, T serotypes 4 and 11.

The molecular mechanism of high level tetracycline resistance in T serotypes 4 and 11 group A streptococcal (GAS) isolates was examined in 61 tetracycline-resistant isolates in Japan. PCR and sequencing analyses revealed that the T serotype/emm genotype, T4/4 isolates carried tet(O) genes, which were genetically homogenous. The T11/11 and T11/89 isolates carried different subtypes of tet(M) genes, which were present on transposons Tn916 and Tn1545, respectively. In addition, these T11 isolates may have obtained the tet(M) gene after the 1990s, because resistance to tetracycline in T11 isolates was rarely found before then. These results strongly suggested that the T4 and T11 GAS isolates acquired tetracycline-resistance via different molecular mechanisms.

Development of a rapid PCR method using the insertion sequence IS1203 for genotyping Shiga toxin-producing Escherichia coli O157.

We developed a rapid PCR method utilizing the diversity of the insertion site IS1203 for genotyping Shiga toxin-producing Escherichia coli (STEC) O157 (IS1203 PCR typing). DNA fragments digested by PvuII, which cut IS1203 at one site, were ligated with themselves and detected by PCR with outward-facing primer pairs for IS1203. To minimize nonspecific bands, nested PCR was also performed. Two fingerprinting patterns produced from the upstream or downstream regions of IS1203 were obtained within 1 or 2 days. By combining the two patterns, 79 STEC O157 isolates were classified into 39 types, which were then classified into 36 subtypes by pulsed-field gel electrophoresis (PFGE). The discriminatory power of IS1203 PCR typing (D = 0.974) is similar to that of PFGE (D = 0.981). This method can be used for rapid and simplified genotyping.

[Isolation of influenza A H1N2 virus from a returning traveller at Nagoya International Airport].

A reassortant influenza A H1N2 virus was isolated from a returning traveller arriving at Nagoya International Airport, Japan from Indonesia in May, 2002. A Hemagglutination inhibition test revealed that the virus was similar to a vaccine strain of A/NewCaledonia/20/99. A phylogenetic analysis demonstrated that the virus forms a cluster with other influenza A H1N2 viruses isolated in other countries. The reassortment event was theoretically assumed to have occurred between the 1999/2000 and 2000/2001 influenza seasons. Neither A H1N2 nor A H3N1 virus was detected from 256 isolates of AH1 or 177 of AH3 influenza viruses isolated in Aichi Prefecture, Japan between the 1999/2000 and 2001/2002 influenza seasons. This finding suggests the importance of influenza surveillance at an airport quarantine office to detect promptly a novel influenza virus penetrating to Japan.

Identification of a Shiga-toxin type I variant containing an IS1203-like element, from Shiga-toxin producing Escherichia coli O157:H7.

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.

Immunomagnetic capture rt-PCR for detection of norovirus from foods implicated in a foodborne outbreak.

In February 2001, an outbreak of acute gastroenteritis due to Norovirus (NV) occurred among employees of 11 companies in Aichi Prefecture. The illness was strongly associated with eating a delivered box-lunch. The use of magnetic beads coated with the antibody to the baculovirus-expressed recombinant capsid proteins of the Chiba virus (rCV) facilitated capture of NV from the food items implicated in the outbreak. Following immunomagnetic capture, NV bound to the beads was detected by reverse transcriptionpolymerase chain reaction (RT-PCR). Of the nine food items tested, two were positive for a genogroup 1 NV. Sequence analysis of RT-PCR products indicated that the nucleotide sequences of NV strains from foods were almost identical to those of NV strains detected in stool samples of ill patients. As the immunocapture RTPCR method is simple and easy to perform, this technique should be useful for the detection of NV from outbreak-implicated foods.

Isolation and identification of a novel human parechovirus.

A cytopathic agent (A308/99) was isolated using Vero cells from a stool specimen of a 1-year-old patient with transient paralysis. The agent was approximately 28 nm in diameter with a distinct ultrastructure resembling the virus particle of an enterovirus. It could not be neutralized by antisera against human picornaviruses such as human enterovirus, Aichi virus or human parechovirus. The virion contained three capsid proteins with molecular masses of 38, 30.3 and 30 kDa. Determination of the complete nucleotide sequence of A308/99 revealed that the nucleotide and deduced amino acid sequences were closely related to those of human parechoviruses. When 11 regions encoding the structural and non-structural proteins were compared, A308/99 had between 75 and 97 % and 73 and 97 % nucleotide identity with human parechovirus type 1 (HPeV-1) and type 2 (HPeV-2), respectively. The most distinctive divergence was seen in VP1, which had 74.5 % and 73.1 % nucleotide identity with HPeV-1 and HPeV-2, respectively. Viruses related to A308/99 were also isolated from three patients with gastroenteritis, exanthema or respiratory illnesses. A308/99 and these other three isolates had no arginine-glycine-aspartic acid (RGD) motif, which is located near the C terminus of VP1 in HPeV-1 and HPeV-2. A seroepidemiological study revealed that the prevalence of A308/99 antibodies was low (15 %) among infants but became higher with age, reaching more than 80 % by 30 years of age. These observations indicate that A308/99 is genetically close to, but serologically and genetically distinct from, HPeV-1 and HPeV-2 and accordingly can be classified as third serotype of human parechovirus.

Isolation and characterization of a new species of kobuvirus associated with cattle.

A cytopathic agent was isolated using Vero cells from the culture medium of HeLa cells that had been used for more than 30 years in our laboratory. This agent, termed U-1 strain, was serially passed in Vero cells with distinct CPE. Particles of U-1 strain negatively stained with phosphotungstic acid exhibited a distinct surface that resembled Aichi virus. The RNA genome of U-1 strain comprises 8374 nt, with a genome organization analogous to that of picornaviruses. Possible cleavage sites of the large ORF, which encoded a leader protein prior to the capsid protein region, were assigned following amino acid alignment with Aichi virus. The virus sequence had 33 and 75 % amino acid identity with the Aichi virus VP1 and 3D regions, respectively, but no more than 23 and 36 % with those of the prototype strains of other PICORNAVIRIDAE: The dendrogram based on the P1, P2 and P3 proteins indicated that U-1 strain is genetically included in the genus Kobuvirus but is distinct from Aichi virus. Of 72 cattle sera, 43 (59.7 %) were positive for neutralizing antibody against U-1 strain at a titre of 1 : 8 or more. However, sera from 190 humans, 242 monkeys, 139 pigs, 5 horses, 22 dogs and 9 cats did not neutralize U-1 strain at a 1 : 4 dilution. RNA corresponding to U-1 strain was detected in 12 (16.7 %) of 72 faecal samples from cattle by RT-PCR. These results indicated that U-1 strain, suspected to be a contaminant from calf sera, is a new species of the genus Kobuvirus, now termed bovine kobuvirus.

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

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.

[Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001].

Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V. p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001. The percentage of the sporadic diarrhea cases caused by O3:K6 V. p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods. Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases. Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident. Percentage of the outbreaks by O3:K6 V. p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively. From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30). Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V. p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas. Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V. p infection.

Molecular diagnosis of human enteroviruses by phylogeny-based classification by use of the VP4 sequence.

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.