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Viral discovery and sequence recovery using DNA microarrays.

Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL - PLoS Biol. (2003)

Bottom Line: Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery.During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient.This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, USA.

ABSTRACT
Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

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Related in: MedlinePlus

Viral DNA Recovery and Sequencing SchemeHybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced.
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pbio.0000002-g002: Viral DNA Recovery and Sequencing SchemeHybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced.

Mentions: In a parallel approach, we directly recovered hybridized viral sequences from the surface of the microarray. This procedure took advantage of the physical separation achieved during microarray hybridization, which effectively purified the viral nucleic acid from other nucleic acid species present in the sample. Using a tungsten needle, the DNA microarray spot corresponding to the conserved 3′ UTR motif was repeatedly scraped and the hybridized nucleic acid was recovered. This material was subsequently amplified, cloned, and sequenced (Figure 2). The largest clone spanned almost 1.1 kb; this fragment encompassed the 3′ UTR conserved motif and extended into the most 3′ coding region of the viral genome. BLAST analysis revealed 33% identity over 157 amino acids to MHV nucleocapsid, thus confirming the presence of a novel coronavirus (see Figure 1; see Data S1). We subsequently confirmed results obtained from both strategies described above by using a random-primed RT-PCR shotgun sequencing approach that generated contigs totaling approximately 25 kb of viral genome sequence (see Data S1).


Viral discovery and sequence recovery using DNA microarrays.

Wang D, Urisman A, Liu YT, Springer M, Ksiazek TG, Erdman DD, Mardis ER, Hickenbotham M, Magrini V, Eldred J, Latreille JP, Wilson RK, Ganem D, DeRisi JL - PLoS Biol. (2003)

Viral DNA Recovery and Sequencing SchemeHybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC261870&req=5

pbio.0000002-g002: Viral DNA Recovery and Sequencing SchemeHybridized viral sequences were physically scraped from a DNA microarray spot, amplified, cloned, and subsequently sequenced.
Mentions: In a parallel approach, we directly recovered hybridized viral sequences from the surface of the microarray. This procedure took advantage of the physical separation achieved during microarray hybridization, which effectively purified the viral nucleic acid from other nucleic acid species present in the sample. Using a tungsten needle, the DNA microarray spot corresponding to the conserved 3′ UTR motif was repeatedly scraped and the hybridized nucleic acid was recovered. This material was subsequently amplified, cloned, and sequenced (Figure 2). The largest clone spanned almost 1.1 kb; this fragment encompassed the 3′ UTR conserved motif and extended into the most 3′ coding region of the viral genome. BLAST analysis revealed 33% identity over 157 amino acids to MHV nucleocapsid, thus confirming the presence of a novel coronavirus (see Figure 1; see Data S1). We subsequently confirmed results obtained from both strategies described above by using a random-primed RT-PCR shotgun sequencing approach that generated contigs totaling approximately 25 kb of viral genome sequence (see Data S1).

Bottom Line: Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery.During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient.This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, USA.

ABSTRACT
Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.

Show MeSH
Related in: MedlinePlus