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

As members of the genus Enterovirus, the Human enterovirus A and Human enterovirus C virus species comprise most of the species assigned to CV serotype A strains with the exception of serotype A9 [23] . Coxsackievirus serotypes B1-6, coxsackievirus serotype A9 (CVA9), enteroviruses 69 and 73 and the majority of echoviruses are classified within the Human enterovirus B species that is one of the largest species group in the family Picornaviridae [26] . Fig. (2) shows the CLUSTALX analysis of the different coxsackievirus serotype strains used for designing the tiling array. For B1 and B3 strains a consensus sequence was developed based on the sequences available for individual members of these groups, whereas single sequences were used for the strains B4, B5, A16 and A21, due to extensive sequence variations between individual members of these groups [23, 26] . Since sequence diversity among the CV strains was distributed over the entire length of the genome, the first 3000 nucleotides were determined to contain sufficient sequence diversity to identify a strain without ambiguity. Identification of HAV Genotype by Microarray Hybridization. Fig. (3) shows the hybridization profile obtained with a target synthesized by PCR amplification of the plasmid pHAV/7. This plasmid contains a copy of the entire HAV sequence of wild-type HM175 strain HM175 [19, 27] that originated from an Australian outbreak, and was designated as genotype Ib by subsequent sequence analysis [24, 28] . The hybridization signals (normalized probe intensities) produced a profile indicating areas of intense hybridization at the position where the HAV sequences are clustered in the array. However, variations in the intensity of hybridization can be observed within these sequences, where the target hybridization intensities against group 1 probes (hav1Cb) differ from probes derived from groups 2 through 5 (hav2b, hav3b, hav4Cb and hav5Cb) sequences. This is more clearly observed in Fig. (4) , where the normalized probe intensities for individual probes within each group sequence present in the array were converted to average probe intensities and plotted for the target. The plot reveals that the HAV genotype 1b (HM175 wild-type) target hybridized most efficiently to probes from genotype Ib, group 1 consensus sequence (hav1cb). These results are consistent with the fact that the viral genome sequence for HAV HM 175 wt strain (14707) is a member of, and therefore most closely related to, group 1 derived probe sequences. Probes representing a closely related HAV Ib strain from group 2 (hav2b) hybridize the target about two thirds as efficiently, while probes from the more genetically distant genotype II virus (hav3b) hybridize with the least intensity. The other probe groups (hav4cb and 5cb) both representing genotype Ia consensus sequences (Fig. 1 , cluster groups 4 and 5) hybridize less efficiently than genotype Ib. Given the readily observable differences in both the normalized and average signal intensities among the genotype group sequence probes (groups 1-5) following genotype Ib target hybridization, and the fact that viruses belonging to different subgenotypes can differ by as much as 7.5% in sequence [21, 24, 28] , the data in Fig. (4) indicate that it is possible to identify HAV strains at the level of both genotype and subgenotype with this type of array.