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

Abstract

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.

FIG. 1.

Phylogenetic analysis of VP7 nucleotide sequences of G4 and G9 strains. The tree was constructed using the Kimura two-parameter and neighbor-joining methods using MEGA 3.1 software. Bootstrap values are shown at the branch nodes (values of <50% are not shown). The Nicaraguan strains are marked with a bold triangle and the lineages (I) and genotypes (G) are indicated at the right. The number of substitutions per site is indicated by the scale bar. Abbreviations for locations: Aus, Australia; Bel, Belgium; Bra, Brazil; Fin, Finland; Mvd, Montevideo (Uruguay); Nic, Nicaragua; Jap, Japan; Py, Paraguay; SA, South Africa; Swe, Sweden; Tha, Thailand.

FIG. 2.

Unique asparagine insert in G4P[8] virus from 2005 in Nicaragua. Amino acid alignment of the rotavirus VP7 gene using ClustalW, version 1.8, with default parameters. The alignment reveals an asparagine insert at position 76 in the 2005 Nicaraguan isolates, which also is present in isolates from Argentina (1996 to 1998), Uruguay (1999), and Brazil (2000 to 2004). *, conserved residue; :, position with conserved substitutions; ., position with semiconserved substitutions.

FIG. 3.

Amino acid insertions and substitutions affect downstream β-sheets in VP7. Schematic representations of secondary structure predictions, using the PSIPRED protein structure prediction server (8). Both panels depict aa 41 to 160. The residue number is shifted 1 aa in the G4 VP7 sequence (Nic118/05) from position 76 compared to the 2002 strain (NicBH63/02). (Boxes a) Beginning at amino acid 80, the 6-amino-acid β-sheet in the 2002 isolate has mutated into a 7-amino-acid β-sheet in the 2005 isolate. (Boxes b) Located in the vicinity of amino acid 120, the 5-amino-acid β-sheet in the 2002 isolate has evolved into a 6-amino-acid β-sheet in the 2005 isolate. Conf, confirmed; Pred, predicted.

FIG. 4.

Amino acid alignment using ClustalW, version 1.8, with default parameters on the European Bioinformatics Institute server of the rotavirus VP7 gene showing the number of amino acid substitutions in the 2005 Nicaraguan isolates compared to the isolate from 2002. The substitutions occurring at positions A to I are all conserved or semiconserved. The three important antigenic sites (VR-5, aa 86 to 101; VR-7, aa 142 to 152; and VR-8, aa 211 to 223) are underlined. The location of the asparagine insert at position 76 is indicated with a circle. *, conserved residue; :, position with conserved substitutions; ., position with semiconserved substitutions.