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Protocol for metagenomic virus detection in clinical specimens.

Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A - Emerging Infect. Dis. (2015)

Bottom Line: Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population.The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing.We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches.

View Article: PubMed Central - PubMed

ABSTRACT
Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

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Results of comparative next-generation sequencing used for development of tissue tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. A) Sample preparation flowchart to generate 4 next-generation sequencing approaches. B) Results obtained for model tissue (chicken) infected with 4 viruses: vaccinia virus (poxvirus) Sendai virus (paramyxovirus), influenza virus (A/PR8/1934), or reovirus (T3/Bat/G/342/08). The x-axis is log-scaled, and normalized read numbers are indicated. C) Results of marmoset sample proof of principle, Sendai virus–infected lung tissue. The baseline is log-scaled, and normalized read numbers are indicated.
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Figure 8: Results of comparative next-generation sequencing used for development of tissue tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. A) Sample preparation flowchart to generate 4 next-generation sequencing approaches. B) Results obtained for model tissue (chicken) infected with 4 viruses: vaccinia virus (poxvirus) Sendai virus (paramyxovirus), influenza virus (A/PR8/1934), or reovirus (T3/Bat/G/342/08). The x-axis is log-scaled, and normalized read numbers are indicated. C) Results of marmoset sample proof of principle, Sendai virus–infected lung tissue. The baseline is log-scaled, and normalized read numbers are indicated.

Mentions: The TUViD-VM protocol was validated by NGS of 4 aliquots of the model tissue. One aliquot was prepared by using the TUViD-VM protocol developed in this study, and 3 aliquots were prepared by using other approaches commonly used for unbiased virus detection (Figure 8; Technical Appendix). We chose the Invitrogen Life Technologies platform because of its rapid run time and read length, which are crucial for diagnostic purposes. All independent runs were normalized to 1,000,000 output reads for reliable comparison (Table 5; Figure 8). NGS results confirmed the substantial increase in virus nucleic acids, as well as the decrease of host nucleic acids achieved by purification with the novel protocol. The amount of detectable virus nucleic acids was increased >1,000-fold compared with other NGS approaches (Figure 8). For example, although the best NGS approach delivered 40 reads for paramyxovirus in infected chicken tissue, the TUViD-VM protocol resulted in >60,000 reads (97.80% coverage of the complete genome) (Figures 8,9,10; Table 5).


Protocol for metagenomic virus detection in clinical specimens.

Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A - Emerging Infect. Dis. (2015)

Results of comparative next-generation sequencing used for development of tissue tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. A) Sample preparation flowchart to generate 4 next-generation sequencing approaches. B) Results obtained for model tissue (chicken) infected with 4 viruses: vaccinia virus (poxvirus) Sendai virus (paramyxovirus), influenza virus (A/PR8/1934), or reovirus (T3/Bat/G/342/08). The x-axis is log-scaled, and normalized read numbers are indicated. C) Results of marmoset sample proof of principle, Sendai virus–infected lung tissue. The baseline is log-scaled, and normalized read numbers are indicated.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4285256&req=5

Figure 8: Results of comparative next-generation sequencing used for development of tissue tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. A) Sample preparation flowchart to generate 4 next-generation sequencing approaches. B) Results obtained for model tissue (chicken) infected with 4 viruses: vaccinia virus (poxvirus) Sendai virus (paramyxovirus), influenza virus (A/PR8/1934), or reovirus (T3/Bat/G/342/08). The x-axis is log-scaled, and normalized read numbers are indicated. C) Results of marmoset sample proof of principle, Sendai virus–infected lung tissue. The baseline is log-scaled, and normalized read numbers are indicated.
Mentions: The TUViD-VM protocol was validated by NGS of 4 aliquots of the model tissue. One aliquot was prepared by using the TUViD-VM protocol developed in this study, and 3 aliquots were prepared by using other approaches commonly used for unbiased virus detection (Figure 8; Technical Appendix). We chose the Invitrogen Life Technologies platform because of its rapid run time and read length, which are crucial for diagnostic purposes. All independent runs were normalized to 1,000,000 output reads for reliable comparison (Table 5; Figure 8). NGS results confirmed the substantial increase in virus nucleic acids, as well as the decrease of host nucleic acids achieved by purification with the novel protocol. The amount of detectable virus nucleic acids was increased >1,000-fold compared with other NGS approaches (Figure 8). For example, although the best NGS approach delivered 40 reads for paramyxovirus in infected chicken tissue, the TUViD-VM protocol resulted in >60,000 reads (97.80% coverage of the complete genome) (Figures 8,9,10; Table 5).

Bottom Line: Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population.The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing.We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches.

View Article: PubMed Central - PubMed

ABSTRACT
Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

Show MeSH
Related in: MedlinePlus