Additional VP1 and VP2 genomic sequences were determined for two norovirus strains of particular interest: Hu/NoV/KL45/Malaysia/1978/GII.na and Hu/NoV/T091/Tunisia/1976/GII.na. Diagnostic primers, as well as newly designed Brefeldin A clinical trial primers, were used in various combinations to determine a consensus sequence for the entire gene, excepting the GIV.1 norovirus, for which only a partial capsid sequence (360 bp) was obtained (Table S1). GenBank accession numbers for all capsid gene sequences obtained in this study are as follows: “type”:”entrez-nucleotide-range”,”attrs”:”text”:”JN699033-JN699050″,”start_term”:”JN699033″,”end_term”:”JN699050″,”start_term_id”:”393192877″,”end_term_id”:”393192875″JN699033-JN699050 and “type”:”entrez-nucleotide”,”attrs”:”text”:”JN989560″,”term_id”:”379067460″JN989560 (Table S2).

Phylogenetic Analysis of Rotavirus and Norovirus Sequences Nucleotide sequences were aligned using Clustal X 2.1, and alignments were manually edited in MegAlign version 9.0 (Lasergene, Madison, WI) [30]. For maximum likelihood phylogenetic analyses, parameter values for best-fit evolutionary models of nucleotide substitutions were determined using Akaike information criterion (AIC) as implemented in MODELTEST [31]. Phylogenetic trees were inferred by maximum likelihood reconstruction of sequence alignments using PhyML software in the context of evolutionary models [31], [32]. Trees were reconstructed with 500 bootstrap pseudoreplicates for statistical reliability. All trees were visualized and annotated using Fig Tree software (http://tree.bio.ed.ac.uk/software/figtree/).

Nucleotide (nt) and amino acid (aa) variations between selected sequences (percent nt, aa distance) were performed using the Tamura-Nei model and Poisson correction, respectively (MEGA version 5), using multiple sequence alignments generated with Clustal X 2.1 [30], [33]. When determining the non-identical residue variations between a single sequence as compared with the remainder of its phylogenetic cluster, individual sequences or groups of sequences were defined as ��taxa�� and an average distance between groups was calculated. Bayesian evolutionary analyses were performed separately for VP1 gene sequences of GI.1 and GI.3 noroviruses, but not for rotaviruses, as a Bayesian analysis for G9 and G12 rotaviruses has been published recently [8]. Our GI.

3 subset in the analysis contained three sequences obtained from this study (Hu/NoV/B8/CentralAfricanRepublic/1977/GI.3, Hu/NoV/C9/FrenchGuiana/1978/GI.3, and Hu/NoV/C91/FrenchGuiana/1978/GI.3) in addition to sequences available in GenBank, whereas our GI.1 subset contained only GenBank sequences (Table S3). GI.1 norovirus sequences were used as a control, because they belong to Genogroup GI, and the GI.1 sequences available in Batimastat GenBank had a wide-range of collection dates (1968�C2007), similar to those of GI.3 (1977�C2007).