Three microliters of the first PCR products was added to the reaction mixture (final 50 ��l), and the same PCR condition was used for the second-round amplification. DNA sequencing. The PCR products were purified with the QIAquick PCR purification kit and in some cases with a QIAquick gel extraction kit (Qiagen). The purified DNAs more were used as a template for direct sequencing. The sequencing reaction was performed in a 96-well microplate using dye-terminator chemistry with BigDye version 3.1 (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. The reaction products were purified with the Montage SEQ96 sequencing reaction cleanup kit (Millipore, Bedford, MA) and sequenced on an automated ABI 3730 xl DNA analyzer (Applied Biosystems).
To obtain full-length genome sequences, fragment sequences of 0.9, 5.2, and 2.5 from the same individual were aligned at an overlapping region by using the Staden Package (http://staden.sourceforge.net). Phylogenetic analysis. Nucleotide sequences were aligned with an outgroup by using CLUSTAL W version 1.4 (49). A distance matrix of nucleotide substitutions per site was estimated from the alignment according to Kimura’s two-parameter method (23). Neighbor-joining trees (42), maximum-likelihood trees, and UPGMA (unweighted pair-group method with arithmetic averages) trees were generated with 100 bootstrap replicates (10) from the matrix numbers by using MEGA version 3.0 (25). For the phylogenetic analysis of NoV ORF2 complete nucleotide sequences, we included sequences of well-recognized strains identified in the global GII/4 epidemic.
They are the <1996 variants (Lordsdale strain [7], GenBank accession no. "type":"entrez-nucleotide","attrs":"text":"X86557","term_id":"1008952"X86557; Bristol strain [15], accession no. "type":"entrez-nucleotide","attrs":"text":"X76716","term_id":"436410"X76716), the 1995-1996 epidemic variants (Grimsby strain, accession no. "type":"entrez-nucleotide","attrs":"text":"AJ004864","term_id":"4138546"AJ004864; 95/96-US strain [32], accession no. "type":"entrez-nucleotide","attrs":"text":"AF080549","term_id":"5162961"AF080549; Camberwell strain [5], accession no. "type":"entrez-nucleotide","attrs":"text":"U46500","term_id":"1184872"U46500), the 2002-2003 epidemic variants (Farmington Hills strain [53], accession no.
“type”:”entrez-nucleotide”,”attrs”:”text”:”AY502023″,”term_id”:”40950079″AY502023; a United Kingdom strain [6], accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY587990″,”term_id”:”46519755″AY587990), GSK-3 and the 2004-2005 epidemic variants (Hunter strain [3], accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”DQ078794″,”term_id”:”84508502″DQ078794; a Netherlands strain OB2004-083 [46], accession no.