Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in São Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.

Phylogenetic analysis of human group C rotavirus circulating in Brazil reveals a potential unique NSP4 genetic variant and high similarity with Asian strains

Group C rotaviruses (RVC) cause gastroenteritis in humans and animals worldwide, and the evidence for a possible zoonotic role has been recently provided. To gain information on the genetic diversity and relationships between human and animal RVC, we sequenced the VP4, VP7, and NSP4 genes of 12, 19, and 15 human strains, respectively, detected in São Paulo state during historical (1988 and 1993) and recent (2007 and 2008) Brazilian rotavirus surveillance. All RVC strains analyzed in the present study grouped into human genotype (G4-P[2]-E2), and did not show any evidence of animal ancestry. Phylogenetic analysis showed that RVC samples detected in 1988 and 1993 clustered together with strains from distinct continents, indicating that historical RVC strains circulating in São Paulo were closely related to those strains circulating worldwide. All three genes (VP7, VP4 and NSP4) of São Paulo RVC strains isolated in 2007-2008 exhibited close phylogenetic relationship with human RVC strains isolated in China and Japan, suggesting that they are genetically linked, and that a gene flow could be occurring between this Asian countries and Brazil. We identified two distinct clusters in the NSP4 phylogenetic tree. One cluster formed exclusively by human Brazilian strains detected in 1997 and 2003-2004 in Rio de Janeiro, Bahia, and Rio Grande do Sul states (Subgroup II) previously described in a different study, that displayed low sequence identities to other human strains formerly published, and to the Brazilian RVC strains (Subgroup I) characterized in the present study. These data suggests the circulation of two genetic profiles of the NSP4 gene in Brazil. High sequence diversity in NSP4 gene was previously reported in Asia, and additional diversity in NSP4 RVC strains spreading in the world should be expected. More in-depth molecular and epidemiological analysis of human RVC throughout the world will be needed to understand their diversity and clarify their evolution, as well as to develop classifications schemes.

Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil

Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2 percent) sequences clustered with NSP4 genotype B, and 12 sequences (7.8 percent) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.

Molecular Typing of Staphylococcus aureus Isolated from Blood on the Basis of Coagulase Gene Polymorphism and Toxin Genes / 대한진단검사의학회지

BACKGROUND: Coagulase is produced by all strains of Staphylococcus aureus. The 3' coding region of the coagulase (coa) gene contains varying numbers of 81 bp tandem repeats. S. aureus produces a variety of extracellular protein toxins. Here, we typed S. aureus strains isolated from blood by coa gene restriction fragment length polymorphism (RFLP) patterns and toxin gene profiles. METHODS: A total of 120 strains of S. aureus were isolated from blood cultures during 2003-2006 at Kangdong Sacred Heart Hospital. The isolates were typed by PCR RFLP analysis of the coa gene and by multiplex PCR for detection of genes encoding enterotoxins (sea, seb, sec, sed, and see), toxic shock syndrome toxin-1 (tst), exfoliative toxins (eta and etb), mecA and femA. RESULTS: All the S. aureus strains were classified into 16 types on the basis of coa gene RFLP and could be further differentiated into 34 types according to the combined patterns of coa gene RFLP and toxin gene profiles. Of 85 methicillin-resistant S. aureus (MRSA) strains, 43 (50.6%) and 36 (42.4%) belonged to the RFLP pattern L5 and pattern L1, respectively. MRSA strains belonging to pattern L5 frequently carried tst (93.0%) or sec gene (81.4%), and strains belonging to pattern L1 frequently carried sea (88.9%) or see gene (44.4%). The rate of the pattern L5 in MRSA strains increased over the past few years and was higher in intensive care unit than in other wards. CONCLUSIONS: We typed S. aureus strains isolated from blood on the basis of coa gene RFLP and toxin genes. The strains belonging to coa gene RFLP pattern L5 and L1 appeared to be the major types of MRSA isolasted from bacteremia and revealed specific toxin gene profiles according to the coa gene RFLP patterns.

PCR-mediated cloning and expression of the gene for the B-subunit of vibrio cholerae toxin isolated recently in Iran

Knowing the nucleotide sequence of the cholera toxin operon, we designed oligonucleotide primers for its-PCR amplification from local clinical isolates of V. cholerae. The resulting amplification product was cloned in a common pUC18 vector. Subsequently, a part of this operon encoding the cholera toxin B-subunit [CTB] was reamplified and cloned between the BamH 1 and EcoR1 sites of the same vector to create a recombinant plasmid pRI8CTB. Temperature-controlled expression of the target protein was achieved by supplementing pR18CTB with a DNA fragment which contained a strong promoter PR and the gene for a heat-sensitive repressor cI857 of bacteriophage lambda from an expression vector pCQV2. When induced, the constructed plasmid pSCTB18 provided for the production of recombinant CTB secreted into the periplasmic space in a yield of about 3mg per liter of bacterial culture, as revealed by GM 1-ELISA

Clonación y expresión de un gen quimérico de la toxina del Bacillus thuringiensis var. berliner por P.C.R. / Cloning and expression of a chimeric toxingene of Bacillus thuringiensis var. berliner by P.C.R.

El Bacillus thuringiensis (Bt.) vars. berliner y kurstaki, se han usado ampliamente como pesticidas. Estas bacterias producen durante la fase de esporulación una toxina con fuerte actividad lepidoptericida. Los genes que codifican para esas toxinas han sido clonados y expresados recientemente en plantas. Estas plantas transgénicas han mostrado toxicidad por ellas mismas contra los insectos. La metodología conocida como Reacción en Cadena de la Polimerasa (P.C.R.), muy recientemente desarrollada, permite la amplificación de un gen, usando la extensión del ADN por la ADN polimerasa a partir de iniciadores específicos. Un gen truncado conteniendo las 2/3 partes del terminal NH2 del gen original, fue aislado de una preparación de ADN total de esta bacteria usando P.C.R. Este gen quimérico fue acoplado directamente a un promotor inducible de E. coli. El producto del gen sintetizado en E. coli fue identificado por anticuerpos antitoxina y su actividad biológica fue testada usando larvas de Heliothis virescens. El análisis de la secuencia parcial del ADN muestra una alta homología con la secuencia reportada en la región 5', pero tiene una divergencia muy alta en la 3'.