A Microarray Based Approach for the Identification of Common Foodborne Viruses

Ayodeji, Mobolanle; Kulka, Michael; Jackson, Scott A; Patel, Isha; Mammel, Mark; Cebula, Thomas A; Goswami, Biswendu B

Oligonucleotide Arrays. All microarrays used in this study were manufactured by NimbleGen Systems Inc. (Madison, WI) using a maskless array synthesis (MAS) technology for in situ synthesis of DNA oligonucleotides directly onto glass microscopy slides [16, 17] . Oligonucleotide design was based on available complete viral genome sequences obtained from GenBank for CV (n=25), HAV (n=23), Norovirus genogroup I (n=4), Norovirus genogroup II (n=21), rotavirus (various species) segments 3 (n=11), 4 (n=19), 8 (n=11), and 11 (n=12) where n equals the number sequences obtained for each virus group. All genomic sequences within a virus group were aligned using CLUSTALX [22] , and dendrograms were generated and consensus sequences constructed based on these analyses. Examples of these dendrograms are shown for HAV and CV (Figs. 1, 2 , respectively). For the purpose of generating representative viral genomic sequences on which to base subsequent oligonucleotide designs, the HAV strains were clustered into 5 groups whose viral genome sequences were constructed as follows: i) a consensus sequence based on the seven genotype Ib (i.e. genotype I, subgenotype b) strains that clustered into group 1 which includes the HAV HM175/wt strain (M14707), ii) a sequence derived from M20273 based on the pairing of M20273 and AF314208 (genotype Ib sequences in group 2), iii) a sequence derived from the single HAV genotype II sequence (IIb) available (AY032861) and assigned as group 3, iv) two consensus sequences based on either cluster group 4 or 5 derived from fourteen genotype Ia sequences that were clustered into either of these two groups. The three consensus sequences representing cluster groups 1, 4 and 5 were obtained following a group sequence alignment and the assignment of the most frequently occurring nucleotide at positions containing nucleotide differences. The clustering of either one or two sequences within a group (as in groups 3 and 2, respectively) resulted in the selection of a single sequence representing that group. Due to the highly diverse (genetic) nature of the CV genome sequences, clustering of strains for generating a group consensus sequence was only done for serotype strains B1 and B3 (groups 1 and 2, respectively). Four additional unique strain sequences were selected as representative sequences for broadly clustered strains identified as groups 3-6. Viral genomic sequences (approximately 3000 bases) from either the 3' end of the HAV genome group sequences or the 5' end of the CV genome group sequences were submitted for design of a tiling oligonucleotide array consisting of oligonucleotides of length 29, starting at every 5 th base in every sequence, resulting in an overlap of 24 bases in two consecutive oligonucleotides [15] . Similar methods were applied to the development and tiling of oligonucleotides as probes for norovirus and rotavirus sequences on the array. The resulting array contained approximately 13,000 viral probes.