長嶋 茂雄

We studied the role of osteopontin (OPN) in host responses against rotavirus (RV) infection. OPN knockout (OPN-KO) suckling mice were more susceptible to RV (strain EW) infection and showed prolonged diarrhea duration compared to wild-type (WT) suckling mice. OPN in the small intestine of WT mice was expressed after 48 h post-infection. On day 2 postinfection, mRNA levels of interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-15 in OPN-KO mice were lower than in WT mice, although mRNA expression of Th-1- and Th-2-related cytokines in the small intestine were nearly the same between OPN-KO and WT mice. These results suggested that OPN is involved in innate responses against RV infection.

An intrinsic low-level vancomycin resistance (VanC phenotype) in Enterococcus casseliflavus is conferred by either of two subtypes of vanC genes, that is, vanC-2 or vanC-3, which are genetically closely related. To know genetic diversity of vanC-2/C-3 genes among E. casseliflavus, nucleotide sequences of vanC-2/C-3 and other genetic components in vanC gene cluster (vanXYc, vanTc, vanRc, and vanSc) were analyzed for nine clinical isolates and four standard strains that showed low-level vancomycin resistance. While the vanC-2/C-3 gene sequences showed 93-100% identities among the strains examined, two genetic groups were discriminated by phylogenetic analysis: one closely related to the previously reported vanC-2 or vanC-3 genes (vanC-2/C-3 genotype) with 98-100% identity, and the other distinct from the vanC-2/C-3 genotype (93-95% identity). The latter group found in three clinical isolates was considered as a new subtype of vanC and tentatively designated as vanC-4. Between strains with the vanC-2/C-3 genotype and those with vanC-4, vanXYc genes were also genetically discriminated with 92-93% identity. Similar sequence diversity was observed for vanTc, vanRc, and vanSc (88-93% identity). Clonal relatedness among the E. casseliflavus strains was investigated by phylogenetic analysis of atpA gene. While among E. casseliflavus strains with vanC-2/C-3 genotype, extremely high sequence identities of atpA were found (98.7% or higher), these strains showed slightly lower identity to those with vanC-4 (94-96%). These two groups of E. casseliflavus strains were also discriminated by genotyping with arbitrarily primed PCR. These findings indicated that among E. casseliflavus there are at least two genetic lineages with the distinct vanC genes, that is, a single subtype including previously known vanC-2/C-3, and a novel subtype vanC-4.

Prevalence and phylogenetic relatedness of rotaviruses causing diarrheal diseases in children and adults were analyzed in Wuhan, China. During a period between June 2006 and February 2008, group A rotavirus was identified in 24.9% (280/1126) and 7.6% (83/1088) of specimens taken from children and adults, respectively. G3P[8] was the most frequent genotype in both children (66.3%) and adults (62.7%), followed by G1P[8] (20.3% and 26.2%, respectively). G9 was detected in specimens from six children (2.0%) and seven adults (5.6%). The VP7 genes of G3P[8] rotaviruses from children and adults showed extremely high sequence identities to each other (98.9-100%) and also to those of G3 viruses isolated in Wuhan in 2003-2004. In the phylogenetic analysis of the VP7 gene, the G3P[8] rotaviruses in Wuhan were clustered into a single lineage with some G3 viruses, which had been referred to as "the new variant G3" rotaviruses, reported recently in East Asia and Southeast Asia. Similar to G3P[8] rotaviruses, extremely high sequence identities between children and adults were observed for VP7 genes of G1 and G9 rotaviruses. The G9 viruses were clustered in the lineage of globally spreading strains, while G1 viruses were genetically close to those reported previously in China and Japan. These findings indicated the persistence of the variant G3 rotaviruses and spread of G9 rotaviruses derived from the global G9 lineage in Wuhan, and suggested that the rotaviruses were circulating among children and adults, irrelevant to the G types. J. Med. Virol. 81:382-389, 2009. (c) 2008Wiley-Liss, Inc.

Novel rotavirus strains B219 and ADRV-N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3. In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined. The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes. The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV-N (strain J19) sequences (87.7-94.3% and 88.7-98.7%, respectively). In contrast, sequence identity to groups A-C rotavirus genes was less than 61%. However, functionally important domains and structural characteristics in VP1-VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins. Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups.

To clarify the phylogenetic relatedness of rotaviruses causing gastroenteritis in children and adults, an epidemiologic investigation was conducted in Mymensingh, Bangladesh, during the period between July 2004 and June 2006. A total of 2,540 stool specimens from diarrheal patients from three hospitals were analyzed. Overall, rotavirus-positive rates in children and adults were 26.4 and 10.1%, respectively. Among the 155 rotavirus specimens examined genetically from both children and adults, the most frequent G genotype was G2 (detection rate: 54.0 and 47.6%, respectively), followed by G1 (21.2 and 26.2%, respectively), and G9 (15.9 and 9.5%, respectively). G12 was also detected in five specimens (3.2% in total; four children and one adult). Sequence identities of VP7 genes of G2 rotaviruses from children and adults were higher than 97.8%, while these Bangladeshi G2 viruses showed generally lower identities to G2 rotaviruses reported elsewhere in the world, except for some strains reported in African countries. Similarly, extremely high sequence identities between children and adults were observed for VP7 genes of G1, G9 and G12 rotaviruses, and also for the VP4 genes of P[4], P[6], and P[8] viruses. Rotaviruses from children and adults detected in this study were included in a single cluster in phylogenetic dendrograms of VP7 or VP4 genes of individual G/P types. Rotaviruses with two emerging types, G9 and G12, had VP7 genes that were phylogenetically close to those of individual G-types recently reported in Bangladesh and India and were included in the globally spreading lineages of these G-types. These findings suggested that genetically identical rotaviruses, including those with the emerging types G9 and G12, were circulating among children and adults in city and rural areas of Bangladesh.

To assess the spread and genetic characteristics of Panton-Valentine leukocidin (PVL) gene-carrying Staphylococcus aureus in Bangladesh, we investigated 59 strains (49 isolates from clinical specimens and 10 isolates colonized in the nasal cavities of medical staff), including 26 methicillin-resistant S. aureus (MRSA) strains. The PVL gene was detected only in methicillin-susceptible S. aureus (MSSA) strains (7 clinical strains and 2 colonizing strains). PVL gene-positive MSSA strains were found to belong to coagulase serotypes III or VI and were classified into sequence types ST88 (CC88), ST772, and ST573 (CC1) by multilocus sequence typing, and agr types 2 or 3. These types were different from those determined for MRSA (coagulase scrotypes I and IV, ST240 and ST361, and agr type 1). PVL gene-positive MSSA possessed a larger number of virulence factor genes than MRSA, although they were susceptible to more antimicrobials. These findings Suggest that the PVL gene is distributed to limited populations of S. aureus clones with specific genetic traits that are distinct from MRSA in Bangladesh, but genetically close to CA-MRSA clones in the CC1 lineage reported in the United States and European countries.

Secondary structural elements at the 5' end of picornavirus genomic RNA function as cis-acting replication elements and are known to interact specifically with viral P3 proteins in several picornaviruses. In poliovirus, ribonucleoprotein complex formation at the 5' end of the genome is required for negative-strand synthesis. We have previously shown that the 5'-end 115 nucleotides of the Aichi virus genome, which are predicted to fold into two stem-loops (SL-A and SL-C) and one pseudoknot (PK-B), act as a cis-acting replication element and that correct folding of these structures is required for negative-strand synthesis. In this study, we investigated the interaction between the 5'-terminal 120 nucleotides of the genome and the P3 proteins, 3AB, 3ABC, 3C, and 3CD, by gel shift assay and Northwestern analysis. The results showed that 3ABC and 3CD bound to the 5'-terminal region specifically. The binding of 3ABC was observed on both assays, while that of 3CD was detected only on Northwestern analysis. No binding of 3AB or K was observed. Binding assays using mutant RNAs demonstrated that disruption of the base pairings of the stem of SL-A and one of the two stem segments of PK-B (stem-B1) abolished the 3ABC binding. In addition, the specific nucleotide sequence of stem-B1 was responsible for the efficient 3ABC binding. These results suggest that the interaction of 3ABC with the 5'-terminal region of the genome is involved in negative-strand synthesis. On the other hand, the ability of 3CD to interact with the 5'-terminal region did not correlate with the RNA replication ability.

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5′ end) formed at the 5′ end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5′-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5′-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5′-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5′-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5′ end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3′ end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5′ end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aichi virus is a member of the family Picornaviridae. Computer-assisted secondary structure prediction suggested the formation of three stem-loop structures (SL-A, SL-B, and SL-C from the 5′ end) within the 5′-end 120 nucleotides of the genome. We have already shown that the most 5′-end stem-loop, SL-A, is critical for viral RNA replication. Here, using an infectious cDNA clone and a replicon harboring a luciferase gene, we revealed that formation of SL-B and SL-C on the positive strand is essential for viral RNA replication. In addition, the specific nucleotide sequence of the loop segment of SL-B was also shown to be critical for viral RNA replication. Mutations of the upper and lower stems of SL-C that do not disrupt the base-pairings hardly affected RNA replication, but decreased the yields of viable viruses significantly compared with for the wild-type. This suggests that SL-C plays a role at some step besides RNA replication during virus infection. © 2003 Elsevier Science (USA). All rights reserved.