key: cord-0034798-wzb32zrm
authors: Wise, Annabel G.; Kiupel, Matti; Maes, Roger K.
title: Genotype-Specific Detection of Ferret Coronavirus by Conventional and Real-Time Reverse Transcription Polymerase Chain Reaction
date: 2015-09-10
journal: Animal Coronaviruses
DOI: 10.1007/978-1-4939-3414-0_16
sha: 50c83c5c1980f629d760752663f42f5497a0bf76
doc_id: 34798
cord_uid: wzb32zrm

Ferret coronavirus is associated with two disease presentations in ferrets, namely, epizootic catarrhal enteritis and a feline infectious peritonitis (FIP)-like systemic disease. In this chapter, we describe conventional and real-time one-step reverse transcription polymerase chain reaction assays that are routinely used in our laboratory to detect either genotype 1 or genotype 2 ferret coronavirus in clinical specimens. These assays were designed based upon the conserved spike gene sequence difference found between three strains of ferret systemic coronavirus and three strains of ferret enteric coronavirus. Recent literature evidence indicates that pathotype is not associated with a specific genotype, and therefore, it is important to test for both genotypes either in enteric or systemic disease.

Epizootic catarrhal enteritis (ECE), an enteric disease of domestic ferrets was fi rst described in the USA in 1993 [ 1 ] . Clinically, the disease is characterized by a foul-smelling bright green diarrhea with high mucus content, lethargy, anorexia, and vomiting. Morbidity approaches 100 %, but mortality is usually below 5 %, with juvenile ferrets often developing only mild disease. ECE was found to be associated with an alphacoronavirus, designated ferret enteric coronavirus (FRECV) [ 2 ] . This disease or the enteric coronavirus agent associated with it has since spread in the USA and other countries worldwide [ 3 -5 ] . Close to a decade after the recognition of ECE, a systemic disease, characterized by feline infectious peritonitis (FIP)-like clinical signs and lesions, began to emerge in ferrets across the USA and Europe [ 6 -9 ] . Clinical fi ndings include anorexia, weight loss, diarrhea and the presence of large palpable intra-abdominal masses. A marked resemblance to FIP is the gross observation of widespread whitish nodules on serosal surfaces and within the parenchyma of abdominal and thoracic organs. A systemic pyogranulomatous infl ammation is the consistent histologic lesion observed in the affected organs, reminiscent of FIP. The agent associated with this disease, was found to be another alphacoronavirus, designated, ferret systemic coronavirus or FRSCV, and is most closely related to FRECV [ 6 , 10 ] . The disease continues to spread worldwide with more recent reports from the UK, France and Japan [ 11 -14 ] .

Comparative sequence analysis of the distal one-third of the genomes of one FRSCV and one FRECV strain [ 10 ] showed that these two viruses share at least 96 % nucleotide sequence identities in the membrane, nucleocapsid and non-structural protein genes, ORFs 3 and 7b. However, their spike (S) proteins showed only 79.6 % amino acid sequence identity. This allowed the development of two S genotype -specifi c conventional [ 10 ] , as well as realtime RT-PCR assays [ 15 ] for routine detection of these viruses in our laboratory. Limited preliminary data have shown that genotype 1 (FRSCV-like) ferret coronaviruses were found associated with the systemic type of illness while genotype 2 (FRECV-like) ferret coronaviruses were detected in cases of ECE [ 10 ] . Recent literature evidence indicates that there is no association between the S genotype and virus pathotype [ 4 , 5 ] . The differentiating genotype-specifi c RT-PCR assays were recently used in a study that determined the prevalence of coronavirus among domestic ferrets in Japan [ 5 ] . The investigators noted that the majority of ferrets that were shedding the genotype 1 ferret coronavirus in feces were not ill with the systemic disease and that some were even asymptomatic. In the Netherlands, partial spike gene sequence analysis reported by Provacia et al. [ 4 ] showed that genotype 1 ferret coronavirus strains were also found present in feces of asymptomatic ferrets. These fi ndings bring to mind the "in vivo mutation" theory proposed by Chang et al. [ 16 ] and Pedersen [ 17 ] for FIPinducing feline coronavirus strains. It may well be the case for ferret coronaviruses that the virus commonly found in normal feces may just be the "precursor" to a more pathogenic strain. Wise et al. [ 10 ] noted the presence of a truncated 3c-like protein gene in two out of three genotype 1 strains identifi ed in ferrets with systemic disease, reminiscent of the 3c gene mutations identifi ed in FIP viruses in cats [ 16 -18 ] .

Polymerase chain reaction ( PCR ) is a method to amplify a fragment of double-stranded DNA to millions of copies, based upon repeated cycles of double-stranded DNA template denaturation, primer annealing and elongation. A primer is a short singlestranded DNA sequence, also called an oligonucleotide, that "primes" the reaction at the appropriate annealing temperature by binding to the complementary sequence of the denatured DNA target. Using the deoxyribonucleotides (dNTPs) in the reaction, the Taq polymerase enzyme then catalyzes the polymerization of nucleotides to polynucleotides beginning at the 3′ end of the primer in the elongation step. Reverse transcription polymerase chain reaction ( RT-PCR ) is used when the original target template is RNA, such that the RNA is fi rst reverse-transcribed into "copy DNA," or cDNA, by the reverse transcriptase enzyme. The cDNA is then amplifi ed in the PCR step. Conventional PCR/RT-PCR [ 19 -22 ] requires the visualization of the amplifi ed DNA or the PCR product, in an agarose gel matrix submerged in buffer through the process of electrophoresis. Electrophoresis subjects the PCR product to an electric current fl ow in the buffer that causes the DNA to migrate through the gel matrix according to its molecular weight. The PCR product is stained with an intercalating dye that fl uoresces upon exposure to UV light, enabling its visualization alongside a DNA size marker ladder.

In real-time PCR / RT-PCR [ 19 -22 ] , the amplifi ed DNA or the amplicon, is detected and recorded in "real-time" during the assay itself. With a hydrolysis probe-based real-time PCR, also called a "Taqman"-probe based assay, the amplifi cation of DNA in every cycle results to a steady buildup of fl uorescence signal in the reaction that can be detected using a specialized thermocycler with a built-in real-time nucleic acid detection system (e.g., Cepheid SmartCycler system). The probe, an essential component of the reaction mix, is a synthetic oligonucleotide with a sequence complementary to the target DNA fl anked by the primers. The probe is labeled at its 5′ end with a fl uorescent reporter dye (e.g., FAM) and with a fl uorescence quencher (e.g., BHQ-1) at its 3′ end. There is no fl uorescent signal emitted as long as the probe remains intact. In the presence of the specifi c target DNA in the reaction, after the primers and probe anneal to their complementary sequence, polymerization takes place in the elongation phase of the PCR cycle, during which the probe is degraded via the 5′ exonuclease activity of the Taq DNA polymerase. Probe degradation leads to the release of the reporter dye from the probe and from its proximity to the quencher molecule, resulting to the accumulation of fl uorescence signal in the reaction mix at every PCR cycle.

1. QIAGEN RNeasy Mini Kit.

2. β-mercaptoethanol (β-ME).

3. Ethanol (EtOH), 96-100 % grade. 4 . 70 % EtOH (prepared by adding three parts molecular grade water to seven parts 100 % EtOH).

5. RNase/DNase-free water (molecular grade water). 6. RNase/DNase-free water (negative control).

7. Micropipettes, fi ltered tips, sterile RNAse-free microcentrifuge tubes, 0.2 ml PCR tubes, and PCR tube rack.

8. PCR tube mini-centrifuge. 9 . Thermocycler (ABI 2720, Applied Biosystems).

1. 50× TAE (Tris-acetate-EDTA) buffer.

2. Agarose powder.

3. 100 bp DNA Ladder (New England Biolabs).

4. 6× gel loading buffer (this comes with the DNA ladder).

5. Ethidium bromide, 10 mg/ml.

6. Gel casting tray (for 100 ml gel volume) and comb (10-slot comb) ( see Note 2 ).

7. Agarose gel electrophoresis apparatus.

8. Power supply.

9. UV light box/transilluminator; UV protective face shield.

10. Gel photography equipment.

1. AgPath ID One-Step RT-PCR Kit (Applied Biosystems, ThermoFisher).

2. Genotype 1-specifi c (G1rt) primers and probe ( see Notes 1 and 3 ). 

( see Note 6 ).

(a) Add 10 μl of β-ME ( see Note 7 ) for every 10 ml of buffer RLT (lysis buffer) ( see Note 8 ). This lysis buffer is stable at room temperature for 1 month after the addition of β-ME.

(supplied as a concentrate), according to the manufacturer's instructions as indicated on the bottle to obtain a working solution.

2. Sample preparation ( see Note 9 ):

(a) Prepare ~10 % fecal homogenate by diluting feces in 0.1 M PBS. Clarify the sample by centrifuging for 5 min at 2500 rpm (~580 rcf for a 24-well microcentrifuge (e.g. Eppendorf Centrifuge 5417C)).

(b) For tissues, prepare ~10 % tissue homogenate in 0.1 M PBS ( see Note 10 ).

3. Pipet 140 μl of sample into a microcentrifuge tube.

4. Add 560 μl of buffer RLT with β-ME to the sample. Mix gently by pipetting.

5. Add 700 μl of 70 % EtOH to the lysed sample. Mix gently by pipetting.

6. Transfer 700 μl of the sample solution to an RNeasy mini column supplied with a 2-ml fi ltrate collection tube.

7. Close the column and centrifuge for 30 s at 10,000 rpm. Discard the fi ltrate and reuse the collection tube in step 8.

8. Transfer the remaining sample solution (~700 μl) to the same RNeasy mini column and centrifuge for 30 s at 10,000 rpm. Discard the fi ltrate and reuse the collection tube in step 9.

9. Add 700 μl of buffer RW1 ( see Note 11 ) to the RNeasy mini column. Close the column and centrifuge for 30 s at 10,000 rpm. Discard the fi ltrate and the collection tube.

10. Place the RNeasy mini column into a new 2 ml collection tube. Add 500 μl of buffer RPE (previously reconstituted with EtOH) to the column for a fi rst wash. Close the column and centrifuge the column for 30 s at 10,000 rpm. Discard the fi ltrate and reuse the collection tube in step 11.

11. Add another 500 μl of buffer RPE to the RNeasy column for the second wash. Close the column and centrifuge for 2 min at 10,000 rpm. Discard the fi ltrate and collection tube.

12. Place the RNeasy mini column with the cap closed into a new 2 ml collection tube. Dry spin the column for 1 min at 14,000 rpm. Residual RPE buffer will be removed from the column ( see Note 12 ). Discard the collection tube.

13. To elute the RNA, place the RNeasy mini column into a new microcentrifuge tube. Pipet 100 μl of RNase/DNase-free water into the column close to the silica-gel membrane. Close the column, let stand for 1 min at room temperature, then centrifuge for 1 min at 10,000 rpm. Discard the column.

14. Put the microcentrifuge tube with the extracted RNA on ice ( see Note 13 ). The RNA sample is now ready to be tested.

Store the RNA in refrigeration temperature if testing within 24 h. Store it in −20 or −80 °C if testing will be delayed for more than 24 h.

1. Working in a designated "clean" PCR station ( see Note 14 ), thaw the OneStep RT-PCR kit reagents and working stock primers on ice. In a sterile microcentrifuge tube, prepare the master mix for the number of reactions needed per genotype test according to Tables 1 and 2 . Total number of reactions ( N ) equals the number of samples to be tested, plus two more for positive and negative controls, then add to this subtotal 10 % more reactions for overage allowance. 6. Centrifuge the tubes briefl y.

7. Place the tubes in the thermocycler and run the RT-PCR reaction according to the cycling protocol shown in Table 3 .

8. When the run is completed, analyze the PCR products by agarose gel electrophoresis . The products may be kept in the refrigerator or frozen for short-term holding.

1. Preparing 1 l (1000 ml) of 1× TAE buffer from a 50× TAE stock solution:

(a) Pour 20 ml of 50× concentrated TAE buffer into a 1 l graduated cylinder.

(b) Fill the cylinder with deionized water up to the 1 l line, making a 1× fi nal concentration of TAE buffer.

2. Preparing a working stock solution of 100 bp DNA ladder with pre-added loading dye: Table 3 Cycling conditions for RT-PCR

Step Temperature (°C) Time 11. When DNA migration is complete, turn off the power supply. Unplug the leads from the gel tank, lift the cover and remove the gel tray.

12. Set the gel on top of the UV transilluminator. Turn off the room light.

13. While wearing a UV protective face shield, turn the UV light on to visualize the gel.

14. The expected product sizes are: 157 bp for genotype 1 and 146 bp for genotype 2 ferret coronavirus (Fig. 1a, b ) . 15 . Document the results with the photo-documentation instrument. Turn the UV light off when fi nished. 2. Click on the SmartCycler software icon to start the program. If using only one SmartCycler block, it will be designated as block "A". The lighted "0" will switch to the letter designation ( see Note 22 ). This confi rms that the computer has established connection with the instrument.

3. Click "Defi ne Graphs" located at the toolbar on the top of the screen ( see Note 23 ).

(a) Select "FAM" under the Graph list, then select or check the box for the following options: Check "Automatically added to new Runs"; for "Graph Type" select Optics; for "Channels" select Ch 1; under "Show" check Primary Curve, Threshold (Horizontal) and Threshold Crossings (Vertical); for "Axes" select Fluorescence vs. Cycle. Click "Save Graph".

(b) Select "Temperature" under the Graph list. Then select or check the box for "Sample Temperature". Check "Automatically added to new Runs". Click "Save Graph".

4. Click "Defi ne Protocols" located in the tool bar at the top of the screen. Click "New Protocol" at the bottom of the screen, then type a "Protocol Name" (e.g., Ferret Coronavirus S Genotyping ) and enter the cycling protocol parameters according to Table 4 . Save the protocol. 5 . Click "Create a Run". In defi ning a protocol in the SmartCycler, enter these steps as "Stage 2 with 3-Temperature Cycle". Enter "1.6" under Deg/Sec column 6. Assign a "Run Name".

7. Leave the Dye Set at the default setting, "FCTC25."

8. Click "Select Graphs" at the bottom of the screen. Under "All Graphs" on the left side of the screen, highlight "FAM" and "Temperature", then click the right-pointing arrow to transfer these graphs under "Selected Graphs" on the right. Click "OK". Unused graphs under "Selected Graphs", if present, may be deselected by highlighting the graphs then clicking the left-pointing arrow.

9. For Channel/Dye settings, leave FAM at the default Usage setting, "Assay". All other dyes/fl uorophores (Cy3, TxR, and Cy5) may be set to "Unused" ( see Note 24 ).

10. For Analysis Settings, set "Bkgnd Max Cycle" (background maximum cycle) at 28 and "Manual Thresh Fluor Units" (manual threshold fl uorescence units) at 25. Leave the rest of the analysis settings at default.

11. Click "Add Remove Sites". Select the appropriate protocol from the protocol list. Select the "sites" or "wells" to be used (e.g., A1, A2, A3) depending on the number of samples to be run, plus two more sites for the positive and the negative controls.

12. Click the arrowhead symbol to add the sites to the selections column on the right. Click the "OK" button. IDs in the table. 14. Prepare the RT-PCR master mix according to Tables 5 or 6 . 15. On a chilled SmartCycler reaction tube rack ( see Note 25 ), put the appropriate number of SmartCycler reaction tubes needed Table 5 Components 20. When the run is fi nished, the Results Table will show for each Site ID whether the sample is "positive" or "negative". Positive samples, including the positive control, will have a FAM Ct (cycle threshold) > 0. Negative samples, including the negative control, will have a FAM Ct = 0.00 ( see Note 26 ).

21. These tabulated results should be confi rmed by viewing the amplifi cation curve, also known as growth curve, for each sample and control, by selecting "FAM" under the "Views" pane. A sample is truly positive when the plot of its fl uorescence vs. cycle number produces a sigmoidal shaped amplifi cation curve as it crosses the threshold setting and continues to generate increasing fl uorescent signal until it plateaus or reaches the last cycle (Fig. 2 ) . The amplifi cation graph of a negative sample and the negative control will appear as a fl at line (Fig. 2 ) ( see Note 26 ). This is the 10× probe stock. To obtain a 10 μM 1× working probe stock, dilute 5 μl of the 10× stock solution in 45 μl of RNAse/DNAse-free water. Use amber-colored tubes for the working stocks and store probes at −20 °C or colder.

5. G2 rt primers have the same sequences as the G2 primers for conventional RT-PCR .

6. Refer to the RNeasy Mini Handbook for description of the RNeasy principle/technology, additional chemical safety information (e.g., how to obtain material safety data sheets online), troubleshooting tips, and general remarks on handling RNA.

7. β-ME is a toxic chemical that must be dispensed in a chemical fume hood. Always handle with gloves, protective clothing and eyewear (personal protective gear).

8. Buffer RLT contains guanidine thiocyanate. It is harmful by inhalation, in contact with skin, and if swallowed.

9. When handling fresh animal tissues and feces , personal protective gear must be worn at all times and work must take place in Biosafety Level (BSL) 2 containment using a BSL 2 hood with the HEPA-fi lter blower on. The blower may be turned off once the specimens are inactivated in the RLT lysis buffer.

10. For animals exhibiting FIP-like lesions, collect sections of grossly affected tissues (i.e., mesenteric adipose tissue and lymph nodes, lung, liver, and spleen). Tissue homogenization is optimally performed by rapid agitation in the presence of a bead or beads using a laboratory mixer mill. Alternatively (if a mixer mill is not available), 25 mg of tissue can be directly disrupted and homogenized in 750 μl of buffer RLT with β-ME in a microcentrifuge tube using a disposable RNAse-free pestle. The tube is then centrifuged for 5 min at 5000 rpm (~2300 rcf for a 24-well microcentrifuge (i.e. Eppendorf Centrifuge 5417 C) to clarify the lysate. Transfer 700 μl of the lysate into a fresh tube, then proceed directly to step 5 (addition of 70 % EtOH) to continue with the RNA extraction.

11. Buffer RW1 contains ethanol and therefore is fl ammable.

12. Removal of residual RPE buffer in this step is crucial. The wash buffer contains EtOH which is inhibitory to PCR .

13. Holding RNA samples at 4 °C or below prior to running the RT-PCR is very important to keep its integrity. This minimizes the chance of any trace contamination with ribonucleases (RNase enzymes) in the sample to be active and degrade the RNA.

14. To minimize exposure of a PCR reaction to RNA/ DNA contamination that lead to false positives, it is optimal to designate a "clean" room where master mixes can be prepared separate from where nucleic acids are extracted and PCR templates added. Additionally designate "clean" pipettes for use solely for master mix preparation and dispensing, and negative control addition.

15. The purpose of the negative control is to monitor the integrity of the master mix, ascertaining that it remains free of contaminating positive PCR template (no amplicon observed in the gel) for the run to be valid. 16 . It is best to use a positive control RNA template diluted sufficiently to a working concentration that produces only a moderately strong band/amplicon in the gel. This will minimize the production of large amounts of positive PCR products that build up over time in the environment and often lead to PCR contamination.

17. Take care that the agarose solution does not boil over while melting it in the microwave. To prevent this from occurring, the microwave should be turned off immediately as bubbling starts to occur, the fl ask taken out and swirled for even mixing of the solution, then heated again in the microwave until the agarose is completely melted. Protective mitts must always be used when handling the fl ask with the heated/melted agarose.

18. Ethidium bromide is a mutagenic substance. Always wear personal protective gear when working with this chemical.

19. Before pulling out the combs, make sure that the agarose gel is already fully set. The gel at the bottom of the wells will get torn off as the comb is lifted out if the gel is not yet fully set. Prior to use, always check that the gel is intact at the bottom of every well. A hole in the bottom of the well will cause the loaded PCR product to leak out.

20. If using gel combs with smaller teeth, decrease the amount of PCR product + loading dye to be applied, accordingly, to each well, taking care that the product does not overfl ow out of the well. Overfl ow will lead to contamination of the neighboring well which may lead to false positives. 21 . Bromophenol blue, a common loading dye, migrates at a rate similar to that of a 300 bp DNA fragment. 22 . Each SmartCycler II processing block or unit has 16 sites or wells. Each site or well is an independently controlled and programmable I-CORE (Intelligent Cooling/Heating Optical Reaction) module. Up to six blocks may be daisy-chained together and operated with a single computer, mimicking a 96-well reaction set-up. Each block gets designated a letter, from "A" to "F", in a 6-block set-up.

23. Alternatively, a graph may be created from scratch by clicking "New Graph" at the bottom of the screen, assigning a name to the graph (e.g., FAM Amp curve), then selecting the options on the right depending on the purpose of the graph, then click "Save Graph".

24. Changing the "Usage" setting of the nonrelevant dyes, Cy3, TxR, and Cy5, from "Assay" to "Unused" will remove the data columns assigned for these dyes in the Results Table, leaving results displayed only for "FAM", giving a more compact  table of results. 25. To chill a SmartCycler reaction tube rack, we recommend just keeping it in the refrigerator (4 °C) and not in the freezer.

26. The Ct (cycle threshold) value is the PCR cycle number at which the fl uorescence signal emitted by the generated PCR product is above background noise and crosses the manually set threshold line. The higher the amount of the starting target template in the sample, the lower the Ct value. A negative sample or control will not cross the threshold throughout the run and will have a Ct = 0. If the negative control shows a Ct > 0, this indicates that there is template contamination in the master mix, and hence the results of the run are not valid. The contamination issue should be addressed and the run repeated.

Coronavirus-associated epizootic catarrhal enteritis in ferrets

Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets

Occurrence of ferret enteric coronavirus in Brazil (preliminary report)

Enteric coronavirus in ferrets, the Netherlands

Genetic characterization of coronaviruses from domestic ferrets

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret ( Mustelaputorius )

Pathology and immunohistochemistry of a disease resembling feline infectious peritonitis in ferrets ( Mustelaputoriusfuro )

Detection of feline infectious peritonitis virus-like antigen in ferrets

Identifi cation of group 1 coronavirus antigen in multisystemic granulomatous lesions in ferrets ( Musteloputoriusfuro )

Comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus

Systemic coronavirus-associated disease resembling feline infectious peritonitis in ferrets in the UK

Unusual presentation of systemic coronavirus is in a ferret

The fi rst case of feline infectious peritonitis-like pyogranuloma in a ferret infected by coronavirus in Japan

Two cases of systemic coronavirus-associated disease resembling feline infectious peritonitis

Genotype-Specifi c Detection of Ferret Coronavirus in domestic ferrets in Japan

Emerging coronaviruses of ferrets

Feline infectious peritonitis: Insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene

A review of feline enteric coronavirus infection

Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats

Rapid and real-time detection technologies for emerging viruses of biomedical importance

Molecular diagnosis of medical viruses

Update on molecular techniques for diagnostic testing of infectious disease

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings