Protocol for Metagenomic Virus Detection in Clinical Specimens1 - Volume 21, Number 1—January 2015 - Emerging Infectious Disease journal - CDC

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Protocol for Metagenomic Virus Detection in Clinical Specimens1 - Volume 21, Number 1—January 2015 - Emerging Infectious Disease journal - CDC

Volume 21, Number 1—January 2015

Research

Protocol for Metagenomic Virus Detection in Clinical Specimens1

On This Page

• Materials and Methods
• Results
• Discussion
• Suggested Citation

Figures

• Figure 1
• Figure 2
• Figure 3
• Figure 4
• Figure 5
• Figure 6
• Figure 7
• Figure 8
• Figure 9
• Figure 10

Tables

• Table 1
• Table 2
• Table 3
• Table 4
• Table 5

Technical Appendicies

• Technical Appendix

Downloads

• PDF[3.25 MB - 10 pgs]
• RIS[TXT - 2 KB]

Claudia Kohl , Annika Brinkmann, Piotr W. Dabrowski, Aleksandar Radoni��, Andreas Nitsche, and Andreas Kurth

Author affiliations: Robert Koch Institute, Berlin, Germany

Suggested citation for this article

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.

Viruses responsible for disease outbreaks in humans naturally emerge either from the human population or as zoonoses by transmission from animal hosts (1). Viruses can also emerge unnaturally, either directly (e.g., bioterrorist attacks) or accidentally (e.g., laboratory infections). Despite these possibilities of virus emergence, 60% of emerging viruses have a zoonotic origin, thus highlighting transmission from animals to humans as a major threat to public health (2). Whenever viruses emerge, prompt identification of the agent and implementation of control measures to contain the outbreak are required.

Currently, various next-generation sequencing (NGS) approaches provide solutions for detection of purified and concentrated viruses (i.e., from cell culture). However, for clinical specimens, such as blood, other fluids, or infected organ tissues, successful detection of viruses is less likely because virus-to-host genome ratios are insufficient (3–6). Use of tissues from persons with suspected infections for virus detection enables elucidation of infection directly at the site of viral replication. Although detecting viruses directly from infected organ tissue provides obvious and valuable advantages, reliable purification of viruses directly from tissues still remains a challenge.

In this study, we quantifiably and extensively compared classical and modern experimental approaches for virus purification and enrichment to finalize a protocol for unbiased detection of emerging viruses directly from organ tissues (tissue-based unbiased virus detection for viral metagenomics [TUViD-VM]) for an increased signal-to-noise ratio (ratio of virus genome to host genome) in virus detection. Use of this approach will reduce the amount of host nucleic acids required and save money and time in preparation of samples for NGS and the subsequent bioinformatic analysis.

Dr. Kohl is a scientist at the Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Berlin, Germany. Her research interests are the detection of emerging and reemerging viruses and the characterization of novel pathogens.

Acknowledgments

We thank Ute Kramer for providing technical assistance, Brunhilde Schweiger for providing influenza A virus strain PR8/38, Marc Hoferer for providing infected marmoset tissues, and Ina Smith and Ursula Erikli for copyediting this article.

This study was partially supported by a scholarship from the Konrad-Adenauer-Foundation (to C.K).

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Figures

• Figure 1. Schematic description of tissue-based universal virus detection for viral metagenomics protocol. Estimated durations of each step are shown in parentheses. The protocol takes 12 h to complete.
• Figure 2. Validation of test aliquots of infected mode used for development of tissue-based universal virus detection for viral metagenomics protocol. Every ninth aliquot was extracted, and viral copy numbers were...
• Figure 3. Comparison of tissue homogenization methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification:...
• Figure 4. Comparison of filtration methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ...
• Figure 5. Comparison of enrichment methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ...
• Figure 6. Comparison of extraction methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ, relative quantification: RQ...
• Figure 7. Comparison of primers and random amplification methods used for development of tissue-based universal virus detection for viral metagenomics protocol. Copy numbers were measured by quantitative PCR in duplicate. RQ,...
• 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....
• Figure 9. Changes in virus-to-host nucleic acid signal-to-noise ratio during development of tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. Next-generation sequencing results for virus-infected chicken tissue comparatively sequenced were...
• Figure 10. Changes in virus-to-host nucleic acid signal-to-noise ratio during development of tissue-based universal virus detection for viral metagenomics (TUViD-VM) protocol. Next-generation sequencing results for virus-infected marmoset tissue comparatively sequenced were...

Tables

• Table 1. Comparison of methods used to develop a protocol for metagenomic virus detection in infectious disease settings
• Table 2. Sample preparation, infection dose, and copy numbers for virus-infected embryonated chickens after 7 d used to develop a protocol for metagenomic virus detection in infectious disease settings
• Table 3. Properties of 4 viruses used to develop a protocol for metagenomic virus detection in infectious disease settings
• Table 4. PCRs used to develop a protocol for metagenomic virus detection in infectious disease settings
• Table 5. Output of next-generation sequencing for development of a protocol for metagenomic virus detection in infectious disease settings

Technical Appendix

• Technical Appendix.  84 KB

Suggested citation for this article: Kohl C, Brinkmann A, Dabrowski PW, Radonić A, Nitsche A, Kurth A. Protocol for metagenomic virus detection in clinical specimens. Emerg Infect Dis [Internet]. 2015 Jan [date cited]. http://dx.doi.org/10.3201/eid2101.140766

DOI: 10.3201/eid2101.140766

1Preliminary results of this study were presented at the Biodefense and Emerging Infectious Diseases Meeting, January 29, 2014, Washington DC, USA.