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Title: Universal plant virus microarrays, broad spectrum PCR assays, and other tools for virus detection and identification

item Hammond, John

Submitted to: Acta Horticulture Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/19/2009
Publication Date: 8/12/2011
Citation: Hammond, J. 2011. Universal plant virus microarrays, broad spectrum PCR assays, and other tools for virus detection and identification. Acta Horticulture Proceedings. 901:49-60.

Interpretive Summary: Virus infection results in crop losses, so virus detection and identification is critical for the protection of crop yield and quality. Interception at ports of entry, quarantine inspection sites, and plant introduction stations of viruses not yet present within the United States is essential to prevent introduction of new viruses to agricultural and horticultural crops. Sensitive methods for the detection of particular viruses, and some methods for detecting multiple viruses within the same taxonomic grouping are available. However, multiple tests must be carried out in order to have a high probability of detecting all of the viruses known to infect a particular crop, and there is low probability of detection of viruses not previously known to infect the crop. Microarray technology using short synthetic oligonucleotides representing nucleic acid sequences conserved between different members of a viral taxonomic group has been shown effective for detection of mammalian viruses. Indeed, it has been demonstrated that a previously unknown virus could be detected and identified to the viral genus level. The potential for adaption of microarray technology for detection and identification of any plant virus is reviewed, and compared to existing methods for virus detection. This information will be of value to scientists and regulatory officials interested in detection and identification of plant viruses.

Technical Abstract: Microarrays have emerged as an important tool in identifying changes in gene expression under different conditions, including plant responses to pathogen infection. A logical extension of the technology is the detection of the pathogen itself, and a number of laboratories have developed either macroarrays or microarrays capable of detection and differentiation of the viruses known to infect a specific crop. As complete sequences of increasing numbers of viral species become available, and bioinformatics software becomes more advanced, it has become easier to identify regions of sequence that are highly conserved among isolates of an individual viral species, and which can be developed as probes for reliable detection of all isolates. More limited regions of sequence are highly conserved at the viral genus level, and have the potential for use to detect virus species that have not previously been reported or characterized. This possibility has already been exploited with plant viruses through the development of broad spectrum polymerase chain reaction (PCR) assays for a number of virus genera, although the degenerate primers often used in such assays may yield non-specific products from some hosts. However, while it is possible to perform multiplex PCR reactions to detect several distinct viruses in a single assay, the probability of amplifying non-specific products increases significantly as the number of primers in the reaction rises, especially if any of the primers include degeneracies. Multiplexing broad spectrum PCR assays is thus unlikely to prove an effective method for screening for unknowns. Microarrays offer an alternative approach to the challenge of screening a single sample for multiple viruses, and rely on unbiased amplification techniques rather than primers specific to each virus or taxonomic group. The concept of a universal virus detection microarray has been reduced to practice in the field of human virology; an array designed to detect multiple genera of viruses affecting the respiratory tract has been produced. The potential of this microarray to detect a previously unknown virus and characterize it to the genus level was demonstrated by the example of the SARS virus. Because of the hybridization pattern of coronavirus-specific probes reactive with the labeled sample amplification products, the SARS virus was identified as a novel coronavirus. It was further demonstrated that the amplified fragments of the SARS virus trapped by the respiratory virus microarray probes could be recovered from the microarray and sequenced. Several laboratories are now interested in the development in a universal plant virus microarray potentially capable of detecting any plant virus to at least the genus level, and also of differentiating biologically-relevant strains of important viruses. Such a microarray would be able to identify previously unrecognized viruses as members of the characterized virus genera. This ability would be of great value, especially in quarantine and clean stock programs, where interception of unknowns is of paramount importance.