Nobumichi Kobayashi

Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction.

MecA, a structural gene located on the chromosome of Staphylococcus aureus, characterizes methicillin-resistant S. aureus (MRSA), and femA and femB(fem) genes encode proteins which influence the level of methicillin resistance of S. aureus. In order to examine effectiveness of detecting mecA and fem genes in identification of MRSA, the presence of these genes in 237 clinically isolated strains of staphylococci was investigated by polymerase chain reaction (PCR). An amplified mecA DNA fragment of 533 base pairs (bp) was detected in 100% of oxacillin-resistant S. aureus, in 16.7% of oxacillin-sensitive S. aureus, in 81.5% of S. epidermidis, and in 58.3% of other coagulase-negative staphylococci (CNS). While the PCR product of femA (509 bp) or femB (651 bp) was obtained from almost all the S. aureus strains except for five oxacillin-resistant strains (2.5%), neither of these genes were detected in CNS. Therefore, the detection of femA and femB together with mecA by PCR was considered to be a more reliable indicator to identify MRSA by differentiating it from mecA-positive CNS than single detection of mecA.

Whole-genomic analysis of rotavirus strains: current status and future prospects

Studies on genetic diversity of rotaviruses have been primarily based on the genes encoding the antigenically significant VP7 and VP4 proteins. Since the rotavirus genome has 11 segments of RNA that are vulnerable to reassortment events, analyses of the VP7 and VP4 genes may not be sufficient to obtain conclusive data on the overall genetic diversity, or true origin of strains. In the last few years following the advent of the whole-genome-based genotype classification system, the whole genomes of at least 167 human group A rotavirus strains have been analyzed, providing a plethora of new and important information on the complex origin of strains, inter- and intra-genogroup reassortment events, animal-human reassortment events, zoonosis, and genetic linkages involving different group A rotavirus gene segments. In addition, the whole genomes of a limited number of human group B, C and novel group rotavirus strains have been analyzed. This article briefly reviews the available data on whole-genomic analysis of human rotavirus strains. The significance and future prospects of whole-genome-based studies are also discussed.

Emergence of Novel Human Group A Rotavirus G12 Strains in India

ABSTRACT Three rare human G12 strains were detected from diarrheic clinical samples of children (<8 months of age) in Calcutta during a routine surveillance study of rotaviral diarrhea in India. The VP7 genes of G12 strains and their products showed maximum homology (97 to 99% at the nucleotide level and 98% at the amino acid level, respectively) with those of two recently reported G12 strains (from the United States and Thailand) but lesser homology with those of prototype G12 strain L26.

Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4

Summary.Long electropherotype with Subgroup I specificity is a common feature of animal rotaviruses. In an epidemic of infantile gastroenteritis in Manipur, India, long but SG I strains predominated in the outbreak in the year 1987–88. One such strain isolated from that region, following the outbreak had G9P [19] specificity. As this is a rare combination, the gene sequences encoding VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 of this strain were analyzed. All these genes except VP7 were closely related to porcine rotaviruses (95–99% identity at amino acid level) and clustered with the porcine strains in phylogenetic analysis. In addition, it had subgroup I nature and belonged to NSP4 genotype B which is characteristic of animal rotaviruses. This is the first report of a rotavirus with VP6 and NSP4, two crucial proteins thought to be involved in host range restriction and pathogenicity, were of porcine origin and caused diarrhoea in a human host. Among the genes of this strain sequenced so far, only VP7 had highest identity to human strains at amino acid level. This study suggests reassortment may be occurring between human and other animal strains and some of the reassortant viruses may be virulent to humans.

Molecular characterization of a porcine Group A rotavirus strain with G12 genotype specificity

Summary.A porcine Group A rotavirus strain (RU172) was detected and molecularly characterized during a surveillance study conducted for rotavirus infection in a pig farm located in a suburban area of Kolkata City, India. The G12 genotype specificity of RU172 was revealed by PCR-based genotyping assays following addition of a G12 type-specific primer (designed in our laboratory to pick up G12 isolates from field samples) and was confirmed by sequence analysis of the VP7-encoding gene. The RU172 strain exhibited maximum VP7 identities of 93.6% to 94.5% with human G12 strains at the deduced amino acid level. In spite of its G12 genotype nature, RU172 appeared to be distinct from human G12 rotaviruses and, on phylogenetic analysis, formed a separate lineage with human G12 strains. Among the other gene segments analyzed, RU172 belonged to NSP4 genotype B, had a NSP5 and VP6 of porcine origin, and shared maximum VP4 identities with porcine P[7] rotaviruses (94.3%–95.4% at the deduced amino acid level). Therefore, to the best of our knowledge, this is the first report of detection of an animal rotavirus strain with G12 genotype specificity. Detection of strains like RU172 provides vital insights into the genomic diversity of Group A rotaviruses of man and animals.

Species-specific and interspecies relatedness of NSP1 sequences in human, porcine, bovine, feline, and equine rotavirus strains

SummaryWe have sequenced gene 5 encoding NSP1 for three human, two porcine, two bovine, one feline, and five equine rotavirus strains, and compared the nucleotide and deduced amino acid sequences with the published sequences for other various strains. Subgroup I human strains L26, 69M, and DS-1 were found to have a similar NSP1 sequence despite their different G serotypes, VP4 genotypes, and RNA patterns. The NSP1 sequence of the human strain K8 showed a high degree of homology to those of porcine strains OSU and YM. A high degree of homology was found among three equine strains (H2, FI-14, and FI23), but they differed from the other equine strains L338 and H1. The strain H1 was similar to the porcine strains. The feline strain Cat2 showed a high homology to bovine strains UK, RF, and A44. Thus, species-specific and interspecies relatedness of NSP1 sequences among human, porcine, bovine, feline and equine rotaviruses was found. Overall genomic relatedness of strains L26 and YM to various human and animal strains was also examined by RNA-RNA hybridization assay. The present and previous hybridization results showed that there is a good correlation in most strains between overall genomic property (or genogroup) and NSP1 sequence homology.

Human Group B Rotavirus Infections Cause Severe Diarrhea in Children and Adults in Bangladesh

ABSTRACT Human group B rotavirus was detected in 12 of 220 adult patients and 2 of 67 child patients with severe diarrhea in Bangladesh. Group B rotavirus may be virulent in both adults and children, and the virus may be an especially serious diarrheal agent in Bangladesh.

Rotavirus Nonstructural Protein 1 Suppresses Virus-Induced Cellular Apoptosis To Facilitate Viral Growth by Activating the Cell Survival Pathways during Early Stages of Infection

ABSTRACT Following virus infection, one of the cellular responses to limit the virus spread is induction of apoptosis. In the present study, we report role of rotavirus nonstructural protein 1 (NSP1) in regulating apoptosis by activating prosurvival pathways such as phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB (nuclear factor κB) during early hours of infections (2 to 8 hpi). The NSP1 mutant strain A5-16 induces weak and transient activation of Akt (protein kinase B) and p65 NF-κB compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-κB promoter activity or Akt phosphorylation after A5-16 infection could be complemented in cells transfected with plasmid expressing NSP1 after infection with the rotavirus A5-16 strain. In cells either infected with A5-13 or transfected with pcD-NSP1, coimmunoprecipitation of NSP1 with phosphoinositide 3-kinase (PI3K) was observed, indicating that strong activation of PI3K/Akt could be due to its interaction with NSP1. In addition, after infection with same multiplicity of infection, A5-16 showed reduced number of viral particles compared to the A5-13 strain at the end of the replication cycle. A lower growth rate could be due to weak induction of PI3K/Akt and NF-κB, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-κB inhibitors. This effect was interferon independent; however, it was partly due to significantly higher caspase-3 activity, poly-ADP ribose polymerase (PARP) cleavage, and apoptosis during earlier stages of infection with the NSP1 mutant. Thus, our data suggest that NSP1 positively supports rotavirus growth by suppression of premature apoptosis for improved virus growth after infection.

Analysis on Distribution and Genomic Diversity of High-Level Antiseptic Resistance Genes qacA and qacB in Human Clinical Isolates of Staphylococcus aureus

High-level antiseptic resistance of Staphylococcus aureus is mediated by multidrug efflux pumps encoded by qacA and qacB genes. We investigated distribution and genomic diversity of these antiseptic resistance genes in a total of 522 clinical strains of S. aureus isolated recently in a Japanese hospital. The qacA/B gene was detected in 32.6% of methicillin-resistant S. aureus (MRSA) and 7.5% of methicillin-susceptible S. aureus (MSSA), whereas the low-level resistance gene smr, which was examined simultaneously, was detected at lower frequencies in both MRSA (3.3%) and MSSA (5.9%). Epidemiologic typing of S. aureus isolates suggested that higher prevalence of qacA/B in MRSA may be due to spread of a single predominant MRSA strain carrying qacA/B in the hospital. Restriction fragment length polymorphism (RFLP) analysis indicated higher prevalence of the qacB-type gene (59.3%) than the qacA-type gene (40.7%) among the qacA/B genes detected. Nucleotide sequencing analysis revealed the presence of two genetic variants in qacA (V1 and V2) and four variants in qacB (V1-V4) that differ from the qacA prototype in pSK1 by 1-5 nucleotides and 7-9 nucleotides, respectively. Although most strains with qacA-V1, qacA-V2, qacB-V3, and qacB-V4 showed high-level resistance to ethidium bromide (EB)(MIC > 100 microg/ml), all of the S. aureus isolates carrying qacB-V1 and qacB-V2 showed lower MICs of EB and some monovalent cationic antiseptic substances. By analysis of the genomic organization of the qacA/B downstream region, divergent forms of this region rearranged with an insertion of IS256 or IS257 were found primarily for qacB. The downstream region of qacA-V1 was suggested to be an evolutionary origin for other divergent forms. These findings indicated that both qacA and qacB are prevalent in recent clinical isolates, especially in MRSA, and these genes consist of variable genetic variants that may be responsible for different resistance levels against antiseptic substances.

Construction of an Infectious cDNA Clone of Aichi Virus (a New Member of the Family Picornaviridae) and Mutational Analysis of a Stem-Loop Structure at the 5′ End of the Genome

ABSTRACT Aichi virus is the type species of a new genus,Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides was identified at the 5′ end of the genome. Computer-assisted prediction of the secondary structure of the 5′ end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at the 5′ end of the positive strand, but not at the 3′ end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication. In contrast, nucleotide changes in the loop segment did not so severely reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-loop at the 5′ end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.