G12 rotavirus has not been detected anywhere in the world since the first detection of a human strain, L26 (G12, P1B[4]), in the Philippines in 1990. In this study, we isolated a human rotavirus (strain T152) with a VP7 of G12 specificity from the stool of an 11-month-old diarrheic patient in Thailand. The strain T152 exhibited a long RNA pattern and subgroup I specificity. In the comparison of the nucleotide and amino acid sequences of the VP7 gene of strain T152 with those of rotaviruses with different G type specificities, strain T152 showed the highest identity, 90.9 and 93.9%, respectively, to G12 prototype strain L26. In contrast, the VP4 gene of strain T152 showed the highest identity with P[9] specificity of human strains K8 and AU-1 and feline strains Cat2 and FRV-1, with homologies of 89.3 to 90.6% at the nucleotide level and 93.9 to 95.6% at the amino acid level. Thus, strain T152 was found to be a natural reassortant strain with G12 and P[9] specificities.Rotavirus is the major cause of acute gastroenteritis in infants of animals and humans. In developing countries, rotavirus infection results in high mortality, and an annual death rate of 800,000 persons has been estimated (6). Furthermore, in developed countries, rotavirus infection is a cause of high morbidity. However, to date no vaccine has been successful. Rotavirus VP7 and VP4 have independent serotype specificities of the G serotype and P serotype, respectively. A total of 14 G serotypes have been reported. Among them, 10 G serotypes have been detected in humans. G1 to G4 are the major G serotypes, and G5, G6, G8 to G10, and G12 are minor or unusual ones (2, 6). In contrast, 21 P genotypes have been recognized, and at least 10 P genotypes have been detected in humans. Recently, a number of strains with an unusual G or P type and a rare combination of G and P types have been detected in human rotaviruses worldwide (1, 3, 10-12, 16, 17, 25-27).G12 was first detected in stool specimens collected from diarrheic children under 2 years of age between December 1987 and February 1988 in the Philippines (20, 27). In 40 rotavirus-positive stool specimens, 20 samples showed subgroup I and long RNA profile (7). Four samples were adapted to cell culture, and at least two (L26 and L27) of them were found to have G12 and P1B[4] specificities by serological and sequence analyses of their VP4 and VP7 (20,27). Since then, however, no further report on the detection of G12 in humans or animals has been presented, although extensive surveys on the G serotype distribution worldwide have been conducted. In this study, we isolated a human rotavirus with G12 and P[9] specificities in Thailand. MATERIALS AND METHODSStool specimens. A total of 405 stool specimens were collected from diarrheic children in a hospital of the Queen Sirikit National Institute of Child Health, Thailand, between 1998 and 1999. An approximately 10% (wt/vol) stool suspension was prepared in phosphate-buffered saline. For virus isolation in MA-104 cells in roller tube culture, each stool extr...

SUMMARYMecA, 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 offemA 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.

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-B (nuclear factor B) 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-B compared to the isogenic wild-type strain A5-13 in MA104 or HT29 cells. The weak NF-B 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-B, since the A5-13 strain also showed reduced growth in the presence of PI3K or NF-B 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.

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.Rotaviruses are the major cause of acute gastroenteritis in infants and young of a wide variety of mammalian and avian species (11). In developing countries, approximately 130 million infants are infected with rotaviruses and there are 800,000 annual deaths (5, 12). In group A rotaviruses, 15 VP7 G serotypes and 21 VP4 P genotypes have been reported for humans, animals, and birds (17). Though 10 G types and 10 P types from humans have been reported (15), clinically and epidemiologically the most important strains belong to the G1 to G4 serotypes with P[8] and P[4] genotypes (6,14). On the other hand, some unusual types (G5, G8, and G9) and rare combinations of G and P types from different countries have also been reported (1,2,3,4,13,16,20). The human rotavirus G12 strains (L26 and L27) were first detected from Philippines in 1990 (19). After more than a decade, in 2002, human G12 strains Se585 (9) and T152 (15) from the United States and Thailand, respectively, were reported. In this study, we report the detection of three rare G12 strains of human rotaviruses in India.As part of a routine surveillance study for diarrheal diseases, stool samples were collected from children below 4 years of age from B. C. Roy Children's Hospital and also from patients of all age groups admitted to the Infectious Diseases Hospital, Calcutta, India, in 2001. Stool samples were also collected from children with diarrhea from Assam Medical College, Dibrugarh, Assam, India; Capital Hospital and Municipality Hospital, Bhubaneshwar, Orissa, eastern India; and Post-Graduate Institute of Medical Education and Research, Chandigarh, northern India. A total of 454 samples were screened for rotaviruses by RNA electrophoresis as described earlier by Herring et al. (10). The fecal specimens were processed for extraction of rotavirus double-stranded RNA suitable for reverse transcription and amplification of the VP4 and VP7 genes as described previously (4). G and P typing of positive samples was carried out by nested and multiplex PCR with consensus and type-specific primers as described previously (4,7,8,18,21). The amplified products were purified with either the QIAquick gel extraction kit or the PCR purification kit (Qiagen, GmBH) in accordance with the manufacturer's instructions. Direct sequencing was carried out by using ABI PRISM BigDye terminator cycle sequencing kits (Applied Biosystems) with an automated DNA sequencer, the ABI PRISM 310 genetic analyzer (Applied Biosystems). The sequencing of the VP7 genes of all three isolates was repeated three times to...

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.