Protocol for metagenomic virus detection in clinical specimens.

Claudia Kohl,Andreas Kurth,Annika Brinkmann,Aleksandar Radonić,Andreas Nitsche,Piotr W Dabrowski

doi:10.3201/eid2101.140766

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.

Highlights

Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health
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 for an increased signal-tonoise ratio in virus detection
Materials and Methods We first describe how the protocol was developed and evaluated, We describe the final virus purification and enrichment TUViD-VM protocol for metagenomic deep sequencing for nucleic acid from organ tissue (Figure 1)

Summary

Ethics Statement

All procedures regarding the marmoset used in this study were performed in accordance with the European Association of Zoos and Aquaria Husbandry Guidelines for Callitrichidae, 2nd ed., 2010 (http://www.marmosetcare. com/downloads/EAZA_HusbandryGuidelines.pdf), which promotes the highest possible standard for husbandry of zoo animals. All procedures regarding the marmoset used in this study were performed in accordance with the European Association of Zoos and Aquaria Husbandry Guidelines for Callitrichidae, 2nd ed., 2010 All procedures regarding embryonated chicken eggs were based on German Animal Protection Laws. Fertilized chicken eggs at embryonation day 11 were inoculated with virus into the allantois sack or onto the chorioallantoic membrane. Development of embryos was terminated at day 17 of embryonation by cooling the eggs overnight at 4°C. No further specific approval is needed for experiments on embryonated avians before time of hatching. Additional approval from the internal ethics advisory board of the Robert Koch Institute was obtained and is available on request

Study Design

Results

Discussion