Mutated G4P[8] rotavirus associated with a nationwide outbreak of gastroenteritis in Nicaragua in 2005.

During February and March 2005, one of the largest national recorded outbreaks of severe acute gastroenteritis occurred in Nicaragua, affecting >or=64,000 individuals and causing >or=56 deaths, predominantly in children under 5 years of age. Through a nationwide laboratory-based study, stool samples were collected and investigated for rotavirus. Of 108 stool samples examined, 72 (67%) were positive for rotavirus. While 69% (50/72) of the positive samples were found in children less than 2 years of age, 50% (6/12) of the adult samples were positive. A mutated G4P[8] strain was the most commonly recognized strain (85%), followed by mixed G strains (8%) and G9P[8] (7%) strains. Phylogenetic analysis of the VP7 gene revealed that the G4 strains belonged to the emerging lineage Ic and was distantly related to the ST3 and VA70 G4 strains. Secondary structure predictions of the VP7 G4 protein revealed an insert of an asparagine residue in position 76, which, combined with additional mutations, surprisingly modified two downstream beta-sheets at amino acid positions 80 to 85 and 115 to 119. The 2005 G4P[8] strain compared to a G4P[8] strain from 2002 had a substitution of an asparagine residue for threonine (Asn-->Thr) at position 96 within antigenic region A, thus eliminating a potential glycosylation site. The mutated G4 virus was introduced in Nicaragua after 2002 and probably emerged from Brazil, Argentina, or Uruguay.

Ligand-directed labeling of a single lysine residue in hGST A1-1 mutants.

Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.