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

Identification of CV Serotype by Microarray Hybridization. Unlike HAV strains, there is tremendous genetic diversity between CV strains, even within the same species as observed, for example, among serotype B strains although they are all members of HEV species [23, 26] . We sought, therefore, to determine whether this array hybridization technique could be used to identify a CV serotype strain target. A typical hybridization profile with a 746 bp segment amplified from CV strains is shown for CVB1 in Fig. (7, panel A) where the data is presented as average probe intensity for all probes derived from the same group sequence, i.e. probe set. Similar to the results obtained following hybridi-zation with HAV targets, CVB1 targets hybridized very efficiently and with greatest intensity to probes (coxB1Ca) derived from a consensus sequence based on its own sequence, i.e. serotype B1 strains (Fig. 2, group 2) . As indicated by the significantly lower probe intensities, minimal hybridization was observed among the remaining 7 CV probe sets indicating a lower efficiency of hybridization to non-CVB1 sequences represented on the array. In fact, hybridization to probes representing all other (non-CV) viruses was essentially at background signal intensity. The results are consistent with the extensive sequence heterogeneity that exists between the CV serotype A and B virus strains, the members within a serotype (A or B), as well as the probe sets derived from these strains. Importantly, these results demonstrate that even with highly (genetically) diverse viruses, such as coxsackieviruses, this array design can discriminate between strains of the same (or different) virus species. We next sought to determine whether discrimination between virus strains or species was possible when the viral target contains sequences not represented by either an individual or a consensus probe set on the array. To complete this experiment, a 746 bp targets derived from coxsackievirus serotype A3 and A5 strains were hybridized to the array. CVA3 and CVA5 serotype strains are both members of HEA species, however the probes' sequence (group 7, coxA16a) for the species was derived from CVA16 (Fig. 2) . Analysis of normalized probe intensities reveal a striking reduction in the overall level of Fig. (6) . Detection of nucleotide differences between two genetically related HAV strains. Average signal probe intensities were calculated following hybridization of targets generated as PCR products from reverse transcription of RNA derived from HAV HM175 clone 1 infected cells or clarified supernatant from HAV 18f infected cells. The amplified targets (3.7 kb) derived from both clone 1 and 18f contain nucleotide sequences synonymous with the first 2.7 kb (probes 1-543) of the 3.1 kb group 1 (HAV1Cb; Fig. 1 ) consensus sequence used to develop the HAV1Cb probe set that is comprised of 608 probes. The individual points on the graph represent specific probe numbers; however, due to graphical compression of the original data, there are iterative probes not represented by individual points on the graph. Arrows identify the probe number having the peak intensity difference between clone 1 and 18f where a nucleotide(s) present in the consensus sequence is identical to nucleotide(s) in clone 1 but not identical to nucleotide(s) in 18f.