The standard primer sets VP7F/R and Beg9/End9 were used to amplify VP7, VP4F/R and Con2/Con3

to amplify the VP8* subunit of the VP4 gene and 10.1/10.2 to amplify NSP4 [20], [21] and [22]. PCR amplicons were purified using Selleck Galunisertib the QIAquick Gel extraction Kit (Qiagen, Inc., Hilden, Germany) according to the manufacturer’s protocol. Purified cDNA was sequenced using BigDye Sequencing Kit version 3.1 (Applied Biosystems; Foster City, CA, USA) in both directions using the oligonucleotide primer sets used in the gene amplification PCR protocol. The thermal cycling reaction consisted of 30 cycles of 96 °C for 15 s, 50 °C for 10 s and 60 °C for 4 min and the products purified by ethanol precipitation. The nucleotide sequence was determined by Applied Genetic Diagnostics (University of Melbourne, Victoria, Australia), using an ABI 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequence data were analysed utilising the Sequencher® Software program version 4.1 (Gene Codes Corp Inc., Ann Arbor, MI, USA). Sequence identity was determined using the BLAST (Basic Local Alignment Search Tool)

server on the GenBank database at the National Centre for Biotechnology Information, USA (www.ncbi.nlm.nih.gov). Sequences from this study and those obtained from the GenBank MG-132 order database were aligned using ClustalW [23]. Genetic distances were calculated using the Kimura-2 correction parameters at the nucleotide level and phylogenetic trees were constructed using the Neighbor-joining method with 500 bootstrap replicates utilising the program MEGA version 4 [24]. The nucleic acid sequences for genes described in this study have been deposited in GenBank (Accession numbers JN377704-JN377721). For simplicity, samples will be referred to by abbreviate common names, AS07-obV2, AS07-obV12, AS07-obV18, AS07-obV37, AS07-obV42, AS07-obV50,

AS07-obV57, AS07-obV75, AS07-obV93, and AS07-obV121. A total of 107 stool samples were collected during a large gastroenteritis outbreak in Alice Springs (Northern Territory) were sent to the Australian Rotavirus Reference Centre for genotype analysis. Seventy-eight samples were found to be rotavirus positive. through Sixty-five samples were analysed by PAGE and silver stained to allow the visualisation and comparison of the electrophoretic pattern. An RNA electropherotype was visible in 57 samples; all samples displayed an identical long electropherotype (data not shown). Fourteen G9P[8] samples from the outbreak were selected for sequence analysis, including two samples from vaccinated infants. The coding region of the VP7 gene was determined for each of the 14 samples, and revealed a highly conserved gene, which displayed 99.9–100% nucleotide homology and 99.6–100% amino acid identity to each other. No conserved amino acid changes were observed between samples obtained from vaccinated and non-vaccinated patients.