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. The marmoset was kept in Zoo Heidelberg (Heidelberg, Germany) with other marmosets in a species-appropriate environment enriched with material for occupation and activity and adequate feeding regimens 3 times a day. The marmoset that was euthanized did not have any additional signs of illness or infection. The production of specific pathogen–free eggs (VALO BioMedia GmbH, Osterholz-Scharmbeck, Germany) was performed in accordance with guidelines of the European Pharmacopoeia (EP7.0.5.2.2) and the US Department of Agriculture Veterinary Services (Memorandum 800.65).

All procedures regarding embryonated chicken eggs were based on German Animal Protection Laws. For infection, 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. However, additional approval from the internal ethics advisory board of the Robert Koch Institute was obtained and is available on request.

Study Design

To compare classical and modern experimental approaches of virus purification and enrichment, we designed a tissue model for internal organs of chicken, each infected with 1 of 4 viruses (poxvirus [vaccinia virus], reovirus[orthoreovirus], orthomyxovirus [influenza virus], and paramyxovirus [Sendai virus]) at low concentrations (Table 1; Table 2; Technical Appendix). Viruses were chosen on the basis of their role in emerging zoonotic diseases and their morphologic and molecular heterogeneity to obtain results for a broad range of viruses (Table 3).

Model Tissue and Protocol Development

To establish a model tissue, we inoculated specific pathogen–free embryonated chicken eggs with 1 of the prechosen viruses at different concentrations. A detailed description of egg infection and preparation of the model tissue is shown in the Technical Appendix. Reovirus (T3/Bat/Germany/342/08) (11) was chosen to represent a nonenveloped virus, orthomyxovirus (influenza A PR/8/1934) and paramyxovirus (Sendai virus) were chosen to represent enveloped viruses with an RNA genome, and poxvirus (vaccinia virus) was chosen to represent an enveloped virus with a DNA genome (Table 3). Viruses in this study were selected to optimize detection of viral zoonotic emerging diseases and possible virus bioterrorism agents.

Figure 2

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 determined by...

To validate the model tissue homogeneity, we selected every ninth sample for simultaneous RNA/DNA extraction and determined copy numbers for all 4 viruses and the galTBP gene (Figure 2). Samples showed an even Gaussian distribution of virus nucleic acids per aliquot and were considered suitable for subsequent experiments.

To establish a protocol for the purification and detection of unknown viruses from animal tissue, we tested different purification techniques and their combinations, including mechanical, enzymatic, and molecular biological methods; the main aim was to eliminate as much host DNA/RNA and maintain as much virus RNA/DNA as possible to optimize random PCR amplification of unknown viruses. The novel established protocol was tested to detect any virus from lung tissue derived from a New World monkey (marmoset), which had to be euthanized because of the unknown disease-causing agent.

We compared different techniques of virus purification, enrichment, and amplification (detailed description of methods compared is shown in the Technical Appendix). In addition, complex purification techniques (digestion and ultracentrifugation) were compared by conducting experiments that had specific control factors (e.g., ultracentrifugation with different concentrations of sucrose, time and speed) (12). Organization of combinations of different control factors and their variable factors (e.g., concentration levels, duration or speed in orthogonal assays) enables conducting a minimal number of experiments. On the basis of results of all purification techniques, we developed a combined protocol to provide the maximized yield of virus RNA/DNA after purification.

Validation and Analysis of Methods Compared

All compared methods were analyzed simultaneously. Because evaluation of sample quality was ongoing, to exclude any extraction bias, an additional unprocessed control aliquot was extracted and measured with every batch. All results of 1 extraction were rigorously compared with a related control aliquot to normalize any variations caused by extraction, cDNA, and quantitative PCR (qPCR) performance.

Every result was evaluated for increasing the signal-to-noise ratio of virus to host-genome (this ratio is indicated by ). Given that ΔΔx =  Δ measured – Δ control, we assume that the ratio change between virus nucleic acids and host genome is given by ΔΔC_t  = Δ purified – Δ unprocessed, where C_t is the cycle threshold. To visualize relative quantification (RQ), RQ (2 – ΔΔC_t) was plotted against the respective methods. The RQ value indicates the x-fold change compared with that of the control aliquot (e.g., RQ value of 10 means a 10-fold higher Δbetween virus and host genomes compared with the control aliquot) (13). Per definition of the RQ method, the area of significance lays outside RQ values of 0.5 and 2 if the samples show an even Gaussian distribution. Thus, results <0.5 and >2 were considered significant.

An additional scoring system was used to evaluate different methods. For every RQ result that increased the ratio between host and virus nucleic acids, we gave 1 point (maximum +4 points if the method led to better detectability for all 4 viruses), and for every decrease, we subtracted 1 point (minimum is subsequently −4 points). Methods with the highest scores were chosen for establishment of a combined protocol that included purification of unknown viruses from any tissue source (Table 1).

Tissue Homogenate

For homogenization, a small cube of tissue (0.5–1 cm³) was placed in an autoclaved screw-cap tube (Sarstedt, Hildesheim, Germany) containing 1 mL of phosphate-buffered saline (PBS) buffer and 20–30 sterile ceramic beads. Tissue was disrupted by shaking 4 times at maximum speed at intervals of 15 s by using the FastPrep-24 Instrument (MP Biomedicals, Strasbourg, France). The duration of this procedure was ≈0.5 h.

Clearing Centrifugation

A total of 200 μL of homogenate was placed in a 1.5-mL tube and vortexed vigorously. The homogenate was centrifuged for 5 min at 2,000 rpm in a bench top centrifuge (Eppendorf, Hamburg, Germany). The supernatant (≈170 μL) was transferred into a clean tube, and the pellet was discarded. The duration of this procedure was ≈0.25 h.

Ultracentrifugation for Virus Particle Separation

A total of 250 μL of 80% (wt/vol) sucrose solution was pipetted into a 2-3/8″ PA ultracentrifuge tube (Beckman Coulter, Krefeld, Germany) and gently overlayed with ≈3 mL of 20% (wt/vol) sucrose solution. The visibility of the phase interface between the 80% and 20% sucrose solutions was checked. The sucrose solution was gently overlayed with cleared tissue supernatant, and PBS was then added to the tubes. The tubes were centrifuged in an SW60 rotor (Beckman Coulter) at 30,000 rpm for 2 h at 4°C. The duration of this procedure was ≈2 h.

Ultracentrifugation to Pellet Virus Particles

The layer on the interface between the 20% and 80% sucrose solutions was collected and transferred into a 3-1/2″ tube (suitable for Beckmann SW32Ti rotors; Beckman Coulter). The collected layer was resuspended in ≈40 mL of PBS and mixed gently by pipetting up and down. The suspension was centrifuged for 1 h at 20,000 rpm and 4°C. The supernatant was then discarded. The duration of this procedure was ≈1 h. As an alternative method, virus particles can be precipitated overnight by using Peg-It (System Biosciences, Mountain View, CA, USA).

DNA Digestion

The pellet was resuspended in 245 μL of 1× digestion buffer (Turbo DNA Free Kit; Ambion, Darmstadt, Germany). A total of Add 5 μL of Turbo DNase (Turbo DNA Free Kit: Ambion) was added and incubated for 30 min at 37°C. The suspension was transferred to a 1.5-mL reaction tube. A total of 10 μL of stop reagent (Turbo DNA Free Kit; Ambion) wad added, incubated at room temperature for 1 min, and centrifuged at 2,000 rpm for 3 min. The supernatant was transferred to another tube, and pellet was discarded. The duration of this procedure was ≈0.75 h.

Combined TRIzol LS Extraction

A total of 750 μL of TRIzol LS (Invitrogen Life Technologies, Grand Island, NY, USA) was added to ≈250 μL of supernatant from previous procedures and homogenized by pipetting up and down 10 times. The mixture was incubated for 5 min at room temperature and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to precentrifuged phase-lock gel tube (5-Prime, Hilden, Germany). A total of 200 μL of chloroform–isoamyl alcohol was added and mixed by inverting the tube vigorously. The tube was incubated for 15 min at room temperature and centrifuged at 12,000 rpm for 15 min.

Approximately 280 μL of supernatant from the phase-lock gel tube was transferred to another tube containing 1,120 μL of AVL lysis buffer without carrier RNA (Viral RNA Mini Kit; QIAGEN, Hilden, Germany). A total of 700 μL of absolute ethanol was added and mixed by pulse vortexing. The solution was transferred in 600-μL portions to a QIAamp Mini Column, QIAGEN), centrifuged 8,000 rpm for 1 min, and the filtrate was discard. The column was placed in a new collection tube, loaded again, and centrifuged until the lysate was added to the column. A total of 500 μL of 70% (wt/vol) ethanol was added and the column was centrifuged at 8,000 rpm for 3 min.

A mixture of 10 μL of DNase and 70 µL of RDD buffer (RNase-Free DNase Set; QIAGEN) was added to the column and incubated for 15 min at room temperature, as described by the manufacturer. The column was washed with 500 μL of AW1 buffer, centrifuged at 8,000 rpm for 1 min, and the filtrate was discarded. The column was placed in a new tube, 500 μL of AW2 buffer was added, the tube was centrifuged at maximum speed for 3 min, and the filtrate was discarded. The column was then placed in a new tube, and the tube was centrifuged at maximum speed for 1 min to dry the column. A total of 30 μL of elution buffer was added to the column, incubated for 5 min at room temperature, and the column was centrifuged in a new 1.5-mL tube. A total of 30 μL of elution buffer was added to the column, incubated for 5 min at room temperature, and centrifuged in the same tube. RNA (≈60 μL) was chilled on ice. The duration of this procedure was ≈3 h.

Random Amplification

Single-stranded cDNA was produced by using the Reverse Transcription Reagent Kit (Applied Biosystems, Foster City, CA, USA) and adapted for a 50-μL reaction containing 30 μL of RNA, 2 μL (40 μmol/L) of K8N random primer (7), 3.2 μL (25 mmol/L) of dNTPs, 4 μL 10× buffer, 9 μL (50 mmol/L) of MgCl₂, 0.8 μL of RNase inhibitor, 0.6 μL of reverse transcriptase, and 0.4 μL of water). A total of 2 μL of K8N random primers and 3.2 μL of dNTPs were added to the 30 μL of RNA and heated at 95°C for 5 min before quenching on ice. The remaining contents of the mixture were heated at 42°C for 60 min before the enzyme was inactivated at 95°C for 10 min.

Double-stranded cDNA was produced by mixing 2 μL of K8N random primers, 3 μL of Klenow buffer (New England Biolabs, Ipswich, MA, USA), and 2 μL (2.5 mmol/L) of dNTPs with 19 μL of cDNA. The mixture was heated at 95°C for 2 min and cooled to 4°C. A total of 1.67 μL of Klenow fragment (New England Biolabs) was added and the mixture was at 37°C for 60 min. Double-stranded cDNA was purified by using the MSB Spin PCRapace Purification Kit (Invitek, Berlin, Germany) and an elution volume of 30 μL. Random amplification was performed by using the procedures reported by Stang and Korn (7). Successful random amplification (a 200–2,000-bp smear) was visualized by agarose gel electrophoresis of 10 μL of PCR product. The duration of this procedure was ≈4.5 h. Sequence information can be obtained by either cloning into sequencing vectors or by NGS.