Microarray-based detection and genotyping of viral pathogens

Abstract

The detection of viral pathogens is of critical importance in biology, medicine, and agriculture. Unfortunately, existing techniques to screen for a broad spectrum of viruses suffer from severe limitations. To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have developed a genomic strategy for highly parallel viral screening. The cornerstone of this approach is a long oligonucleotide (70-mer) DNA microarray capable of simultaneously detecting hundreds of viruses. Using virally infected cell cultures, we were able to efficiently detect and identify many diverse viruses. Related viral serotypes could be distinguished by the unique pattern of hybridization generated by each virus. Furthermore, by selecting microarray elements derived from highly conserved regions within viral families, individual viruses that were not explicitly represented on the microarray were still detected, raising the possibility that this approach could be used for virus discovery. Finally, by using a random PCR amplification strategy in conjunction with the microarray, we were able to detect multiple viruses in human respiratory specimens without the use of sequence-specific or degenerate primers. This method is versatile and greatly expands the spectrum of detectable viruses in a single assay while simultaneously providing the capability to discriminate among viral subtypes.

Fig 1.

(A) Graphical depiction of homology between coxsackie A21 and other enteroviruses. Picornavirus genomic organization is shown (Left). Seventy-nucleotide segments from the coxsackie A21 genome are ordered sequentially downward from the 5′ end of the genome. The number of nucleotides of identity between each 70-nt segment (rows) and each virus (columns) in the genus enterovirus is reflected by the intensity of the blue bar. Identities of <20 nt were plotted as gray. (B) Homology between RV16 and other RVs. (C) Homology between RV14 and other RVs.

Fig 2.

Detection of multiple viruses by DNA microarray. (A) One small region of a microarray is depicted with its corresponding bar graphic. Each detection oligonucleotide from the microarray is depicted as a vertical stripe. Detection oligonucleotides (stripes) are grouped by viral family of origin, and hybridization intensity on the microarray is reflected by the color of the stripes. Black indicates signal below threshold and Cy5 hybridization intensity above threshold is represented by a continuous color scale (yellow). The upper limit of the linear scale for each microarray was independently set to account for array-to-array hybridization variation. (B) Hybridization results from infection of HeLa cells with adenovirus 12, parainfluenza 3, RSV, polio1, and RV2. Detection oligonucleotides from the herpes and miscellaneous viruses are omitted for clarity (full plots are available in Fig. 5, which is published as supporting information on the PNAS web site, www.pnas.org). (C) Hybridization results from the BCBL-1 cell line, which harbors KSHV. For clarity, only the herpes virus detection oligonucleotides are shown.

Fig 3.

Detection of multiple RV serotypes. Close-up views of the set of RV-derived detection oligonucleotides from hybridizations of different RV-infected HeLa cells. Cy5 hybridization intensities are plotted as a continuous color scale. Oligonucleotides are grouped by serotype of origin, with elements detecting the RV positive strand (+) followed by elements detecting the negative strand (−). The other RV category has 12 pairs of oligonucleotides (forward and reverse complement) derived from miscellaneous RV strains. (A) Four rhinoviruses used in the design of the detection oligonucleotides. (B) Detection of four additional RV serotypes.

Fig 4.

Detection of viruses in isolated nasal lavage. (A) Close-up of RV detection oligonucleotides: (Top) nasal lavage taken from patient before infection with RV16; (Middle) nasal lavage taken 2 days postinfection; (Bottom) amplified RV16-infected HeLa RNA. (B) Close-up of RV detection oligonucleotides from nasal lavage samples M18GV1 and J01LV1. (C) Close-up of family Paramyxoviridae-derived detection oligonucleotides, grouped by individual virus, after hybridization to sample H03AV1. Full plots for each hybridization are available in Fig. 6, which is published as supporting information on the PNAS web site.