High prevalence of human Torque teno virus in streams crossing the city of Manaus, Brazilian Amazon.

AIMS: Torque teno virus (TTV) is a human DNA virus chronically infecting most healthy individuals worldwide and can be transmitted by faecal-oral route. The occurrence of TTV was evaluated in the streams crossing the city of Manaus (Brazilian Amazon) over a 1-year period, four times a year. METHODS AND RESULTS: Fifty-two water samples were collected from 13 different locations. Viruses were concentrated from two litres of water by adsorption to negative membrane filters followed by ultrafiltration. TTV DNA was detected by PCR assays designed to detect all five TTV genomic groups. By conventional PCR, 19/52 (37%) samples were positive. By real-time PCR, TTV DNA could be detected in 48/52 (92%) samples. Viral loads ranged from 1300 to 746 000 genome equivalent per 100 ml of river water. Eleven distinct nucleotide sequences were obtained. CONCLUSIONS: Our results show the wide distribution and diversity of TTV among Manaus urban micro basins. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here may contribute to substantiate TTV as a sensitive indicator of human contamination.

Molecular detection of hepatitis A virus in urban sewage in Rio de Janeiro, Brazil.

AIMS: A one-year survey was conducted to examine hepatitis A virus (HAV) prevalence, distribution of genotypes and their relationship to bacterial indicators in raw and treated sewage samples. METHODS AND RESULTS: Fifty sewage samples (raw = 25 and treated = 25) were collected twice monthly from one sewage treatment plant in Rio de Janeiro. Virus concentration was performed by adsorption to an electronegative membrane followed by ultrafiltration. Viral RNA was detected by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR and positive products were directly sequenced. Total and faecal coliform concentrations were also determined. By nested RT-PCR, HAV RNA was detected in 16/50 (32%) and eight (16%) of them were found in treated sewage samples. By real-time PCR, HAV RNA was detected in 46/50 (92%) samples and 24 were from treated sewage. Phylogenetic analyses classified nine isolates (56%) as subgenotype IA and seven (44%) as IB. CONCLUSIONS: Real-time PCR was more sensitive than nested RT-PCR; the presence of subgenotypes IA and IB was described and bacterial indicators cannot be used to predict HAV presence in sewage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrated that HAV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV infection.

Hepatitis A virus in environmental water samples from the Amazon Basin.

Hepatitis A virus (HAV) is a significant waterborne human pathogen. Of the global supply of potable water, Brazil retains 13%, of which 75% resides in the Amazon Basin. Although hepatitis A morbidity has declined progressively in Brazil as a whole, it remains high in the Amazon region. We used nested and real-time reverse-transcription polymerase chain reaction (RT-PCR) to detect and quantify the viral load in water samples from the Amazon Basin. Most samples tested positive (92%), with viral loads varying from 60 to 5500 copies /L, depending on sanitary conditions and the degree of flooding. Nested RT-PCR of the VP1-2A region detected HAV RNA in 23% of the samples. In low viral load samples, HAV was detected only with real-time RT-PCR, suggesting that this technique is useful for monitoring HAV contamination. The presence of HAV in water samples constitutes a serious public health problem.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37 percent of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89 percent similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Preservation of frozen yeast cells by trehalose.

Two different methods commonly used to preserve intact yeast cells-freezing and freeze-drying-were compared. Different yeast cells submitted to these treatments were stored for 28 days and cell viability assessed during this period. Intact yeast cells showed to be less tolerant to freeze-drying than to freezing. The rate of survival for both treatments could be enhanced by exogenous trehalose (10%) added during freezing and freeze-drying treatments or by a combination of two procedures: a pre-exposure of cells to 40 degrees C for 60 min and addition of trehalose. A maximum survival level of 71.5 +/- 6.3% after freezing could be achieved at the end of a storage period of 28 days, whereas only 25.0 +/- 1.4% showed the ability to tolerate freeze-drying treatment, if both low-temperature treatments were preceded by a heat exposure and addition of trehalose to yeast cells. Increased survival ability was also obtained when the pre-exposure treatment of yeast cells was performed at 10 degrees C for 3 h and trehalose was added: these treatments enhanced cell survival following freezing from 20.5 +/- 7. 7% to 60.0 +/- 3.5%. Although both mild cold and heat shock treatments could enhance cell tolerance to low temperature, only the heat treatment was able to increase the accumulation of intracellular trehalose whereas, during cold shock exposure, the intracellular amount of trehalose remained unaltered. Intracellular trehalose levels seemed not to be the only factor contributing to cell tolerance against freezing and freeze-drying treatments; however, the protection that this sugar confers to cells can be exerted only if it is to be found on both sides of the plasma membrane.