The Separation of Viruses from One Another

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Journal of Chromatography A, 1388 (2015) 69–78

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Journal of Chromatography A

j ournal homepage: www.elsevier.com/locate/chroma

A new application of monolithic supports: The separation of viruses from one another

a,∗ b b a a

J. Rusˇciˇ c´ , I. Gutiérrez-Aguirre , M. Tusekˇ Znidariˇ cˇ , S. Kolundzijaˇ , A. Slana ,

c,1 b a

M. Barut , M. Ravnikar , M. Krajaciˇ c´

Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia

Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia

BIA Separations d.o.o., Ajdovsˇcina,ˇ Slovenia

a r t i c l e i n f o a b s t r a c t

Article history: The emergence of next-generation “deep” sequencing has enabled the study of virus populations with

Received 13 November 2014

much higher resolutions. This new tool increases the possibility of observing mixed infections caused by

Received in revised form 30 January 2015

combinations of plant viruses, which are likely to occur more frequently than previously thought. The bio-

Accepted 30 January 2015

logical impact of co-infecting viruses on their host has yet to be determined and fully understood, and the

Available online 9 February 2015

ﬁrst step towards reaching this goal is the separation and puriﬁcation of individual species. Ion-exchange

monolith chromatography has been used successfully for the puriﬁcation and concentration of different

Keywords:

viruses, and number of them have been separated from plant homogenate or bacterial and eukaryotic

Mixed infections

lysate. Thus, the question remained as to whether different virus species present in a single sample could

Virus strains

be separated. In this study, anion-exchange chromatography using monolithic supports was optimized

Ion-exchange chromatography

Monolithic supports for fast and efﬁcient partial puriﬁcation of three model plant viruses: Turnip yellow mosaic virus, Tomato

Plant viruses bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated

from each other in a single chromatographic experiment from an artiﬁcially mixed sample. Based on

A260/280 ratios, we were able to attribute speciﬁc peaks to a certain viral morphology/structure (icosa-

hedral or rod-shaped). This ﬁrst separation of individual viruses from an artiﬁcially prepared laboratory

mixture should encourage new applications of monolithic chromatographic supports in the separation

of plant, bacterial, or animal viruses from all kinds of mixed samples.

1. Introduction et al. [5] have addressed the advantages and disadvantages of cur-

rent diagnostic techniques.

With further advances in sequencing technology, it is becoming Monolithic supports allowed chromatography to be applied in

apparent that viruses are ubiquitous and that mixed virus infec- virus puriﬁcation and concentration. Unlike classical particle-based

tions are probably a common occurrence [1,2]. Different virus–virus chromatographic supports where mass transfer is based on diffu-

interactions can have variable effects on disease development and sion and pores are relatively small, monoliths are characterized by

can have great economic impact in the case of plant viruses [3], mass transfer greatly enhanced by convection and channels that are

as well as an inﬂuence on human health where human viruses several microns in size [6]. They have been used successfully in virus

are concerned [4]. Furthermore, different strains of the same virus puriﬁcation and concentration from diluted samples [7–18], for

species can have devastating impacts on their hosts, and therefore, monitoring virus titre during bacteriophage [19] and adenovirus-

fast detection and identiﬁcation of mixed infections/different virus based vector production [20], as well as in the puriﬁcation of

strains is of great importance. In a recent review article, Boonham virus-like particles (VLP) and other virus vectors [21–25]. So far,

only two plant viruses, the rod-shaped Tomato mosaic virus (ToMV)

and the ﬁlamentous Potato virus Y (PVY), have been puriﬁed using

monolithic supports, and they were shown to be infective after the

∗ puriﬁcation process [12,17]. Therefore, the ﬁrst aim of this work

Corresponding author at: Marulicev´ trg 9a, 10000 Zagreb, Croatia.

was to extend the range of plant viruses puriﬁed in that way and to

Tel.: +385 1 489 8096.

prove the applicability of this approach by dealing with three new

E-mail address: [email protected] (J. Rusˇciˇ c).´

Present address: Sandoz Biopharmaceuticals, Menges,ˇ Slovenia. models: Turnip yellow mosaic virus (TYMV; Tymovirus, Tymoviridae),

http://dx.doi.org/10.1016/j.chroma.2015.01.097

70 J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78

Table 1

Tomato bushy stunt virus (TBSV; Tombusvirus, Tombusviridae), and

Chromatographic buffers.

Tobacco mosaic virus (TMV; Tobamovirus, Virgaviridae). All three

viruses have pI values under pH 5.0 [26]; therefore, it is reason- Loading buffer Elution buffer

able to assume that at pH > 5.0 they will be negatively charged and

20 mM sodium acetate pH 5.5 20 mM sodium acetate 2 M NaCl pH 5.5

that they will bind to an anion exchanger. 20 mM MES pH 6.0 20 mM MES 2 M NaCl pH 6.0

TYMV and TBSV are small icosahedral stable plant viruses with 20 mM MOPS pH 7.0 20 mM MOPS 2 M NaCl pH 7.0

20 mM HEPES pH 8.0 20 mM HEPES 2 M NaCl pH 8.0

diameters between 30 and 35 nm that reach high titres in exper-

20 mM Tris pH 9.0 20 mM Tris 2 M NaCl pH 9.0

imental hosts and can be puriﬁed in high amounts with classical

puriﬁcation protocols employing ultracentrifugation. They have

had a signiﬁcant role in our understanding of the virus icosa-

Domin. and TMV in N. megalosiphon Van Huerck and Müll. Arg. The

hedral structure [27], both of their detailed structures are known

latter two viruses (TBSV-Cro651, TMV-Cro510) are part of a plant

[28–30], and they are used as models in the research of various

virus collection maintained at the Department of Biology, Univer-

aspects of virus life cycle and cellular pathways [31,32]. The two

sity of Zagreb (Zagreb, Croatia). The infected tissues were collected

viruses are interesting because of their biotechnological applica-

after the onset of virus characteristic symptoms: bright yellow

tions: TYMV-based bionanoparticles have been reported [33–36]

mosaic for TYMV, mottling and leaf distortion for TBSV, and defor-

as well as TBSV-based VLPs [37–39]. TYMV isolates are divided into

mation and vein clearing of the young leaves for TMV. The collected

two strain groups: TYMV-1 and TYMV-2 [40]. The serological rela- ◦

tissues were either frozen at −20 C or immediately homogenized

tion between these two groups is one of the most distant reported

in universal extraction bags with a synthetic intermediate layer

among tymoviruses [41]; strains with that degree of serological

(Bioreba, Reinach, Switzerland) to pre-ﬁlter plant extracts. The

difference with a serological differentiation index (SDI) of 3.5, can

plant tissue mass and homogenizing buffer volume ratio (w/v)

be considered different viruses [41,42]. Nevertheless, because of

was kept at 1/5 throughout the experiments and the homogeniz-

the similarities in their basic chemical composition, host range,

ing buffer was the chromatographic loading buffer used in the

and symptomatology, they are still considered strains of the same

particular experiment. The pre-ﬁltered plant homogenates were

virus. The majority of reported isolates, including the type isolate

centrifuged for 10–15 min at 16,060 × g at room temperature and

that has been thoroughly analyzed, belong to the TYMV-1 group.

the virus containing supernatants were ﬁltered through 0.45 ␮m

We believe that, by investigating the chromatographic properties

HPLC certiﬁed ﬁlters (Spartan 13 mm Syringe Filter, Whatman, GE

of different TYMV isolates, we could establish a basis for their sepa-

Healthcare, Uppsala, Sweden). The virus-containing ﬁltrates were

ration and for monitoring the presence of different strains in mixed ◦

either frozen at −20 C or loaded directly onto the column used in

infections. This would provide a good model for the separation of

the particular experiment. The uninfected plant tissue used in the

strains and isolates of other virus species. Rod-shaped TMV, cer-

chromatographic experiments as a negative control was prepared

tainly the most famous plant virus, was the ﬁrst virus discovered. It

in the same way.

holds the ﬁrst position among the top ten plant viruses in molecular

plant pathology [43] and is still an interesting research model today

as an expression vector in the context of plant-derived vaccines 2.2. Chromatography conditions for optimization and partial

[44]. puriﬁcation

Based on retention times obtained for different bacteriophages,

Adriaenssens et al. [13] and Smrekar et al. [45] suggested that ion- All chromatographic experiments were performed on an Agilent

exchange chromatography on monolithic supports can separate series 1100 (Agilent Technologies, Santa Clara, CA, USA) HPLC sys-

viruses present in the same sample as long as they have different tem. ChemStation software (Agilent Technologies, Santa Clara, CA,

charge properties. The latter group even demonstrated a virtual USA) was used to measure peak area and height for speciﬁc chro-

separation by overlapping chromatograms obtained from separate matographic peaks, and the A260/A280 ratios were calculated. Unless

experiments with individual bacteriophages. To test if this hypoth- otherwise indicated, the reported data was obtained from the peak

esis can be applied to plant viruses as well, we used our three model height values. Two anion exchangers were used in the experiments:

viruses. Furthermore, we included different TYMV isolates/strains Convective Interaction Media (CIM) DEAE and CIM QA (BIA Separa-

in our separation experiments. Our model viruses had different pI tions, Ajdovsˇcina,ˇ Slovenia), with a column volume of 0.34 mL, ID

values: TBSV pI 4.1, TYMV pI 3.7, and TMV pI 3.5 [26]. Therefore, we 12 mm, length 3 mm, 62% porosity, and an average channel size of

assumed that they would have variable degree of net charge and 1.2–1.5 ␮m. Throughout the initial and optimization experiments,

different charge distribution at certain buffer pH values and that several buffers with different pH values were used (Table 1). In all

different amounts of salt would be necessary to elute them from the experiments, the ﬂow rate was set at 2 mL/min and absorbance

the column. Thus, the ultimate aim of this study was to achieve was monitored at 260 nm and 280 nm. Chromatographic runs were

a real separation of viruses. By using ion-exchange chromatogra- performed in triplicate, and fractions were collected manually from

phy on monolithic supports, we were able to separate individual two runs.

plant viruses and virus strains from the laboratory-prepared mixed The TYMV samples, prepared as described in Section 2.1, were

samples. loaded onto a weak anion exchanger (CIM DEAE) or a strong anion

exchanger (CIM QA). Unbound macromolecules were washed out

with 4 mL (12 column volumes) of loading buffer, and the bound

2. Experimental macromolecules were eluted using linear gradient 0–75% elution

buffer in 8 min (47 column volumes). The TBSV and the TMV sam-

2.1. Propagation and preparation of TYMV, TBSV and TMV ples, also prepared as described in Section 2.1, were loaded onto

the CIM DEAE and CIM QA columns, respectively. In both cases,

Several TYMV isolates were propagated in Brassica rapa L var. unbound macromolecules were washed out with 2 mL (6 column

rapa ‘Purple top’ and ‘Turnip Atlantic’. Two isolates, Edinburgh volumes) of loading buffer and the bound macromolecules were

(TYMV-E) and Northumberland (TYMV-N), were obtained by Dr. eluted using linear gradient 0–50% elution buffer in 7 min (41 col-

–

Ðorde Mamula in 1978 from John Innes Centre (Norwich, UK) while umn volumes).

the origin of the third isolate, the Yugoslavia isolate (TYMV-Y), is After each run, the columns were equilibrated with 4–7 mL

described elsewhere [46]. TBSV was propagated in N. benthamiana (12–20 column volumes) of 100% elution buffer followed by 3–4 mL

J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78 71

(9–12 column volumes) of 100% loading buffer. The columns were determined after automatic adjustment of the baseline using SDS

sanitized with 0.5 M NaOH for 15 min at a ﬂow rate of 1 mL/min v1.4 software (Applied Biosystems, Carlsbad, CA, USA). Average Ct

after each change of chromatographic conditions (change of buffer, values for TYMV, TBSV, TMV and LUC were calculated for each frac-

type of sample). The column pH was restored with the appropriate tion and used to estimate TYMV, TBSV and TMV concentrations via

buffer. previously generated standard curves (Fig. S1). Recoveries were cal-

culated as the percentage of virus present in the fractions in relation

2.3. Separation of individual plant viruses from mixed samples to the load [12,48,49]. A buffer control, a cDNA negative control,

and a non-template control (NTC) were included on each plate.

TBSV, TYMV-N, TYMV-E, and TMV partially puriﬁed by chro- For TYMV, samples with Ct > 25–26 were considered to be nega-

matographic methods described in Section 2.2 were used to prepare tive [50]. In the case of TBSV and TMV, samples with Ct > 35 were

various mixed samples. Prior to mixing, chromatographic exper- considered negative because all the negative controls had values

iments were performed with the same amounts of individual that fell into this range. In spite of this, the assays used adequately

viruses; that is, the same volumes used in single runs were also used serve as a tool for virus recovery calculation.

to prepare the mixed samples. This was done in order to determine

whether viruses from a mixed sample had the same retention times

2.7. Transmission electron microscopy (TEM)

as individual viruses. The unbound macromolecules were washed

with 2 mL (6 column volumes) of loading buffer, and the bound

Several elution fractions together with the original extract used

macromolecules were eluted using linear gradient 0–50% elution

for separation were examined under TEM using the negative-

buffer in 7 min (41 column volumes). The column was equilibrated

staining method. Twenty microliters of fractions were applied on

with 4 mL (12 column volumes) of 100% elution buffer followed by

formvar-coated and carbon-stabilized copper grids (400 mesh) at

3 mL (9 column volumes) of 100% loading buffer. Fractions were

room temperature for 5 min and stained with 1% uranyl-acetate

manually collected from duplicate runs. Two sets of conditions

(SPI Supplies, West Chester, PA, USA). The samples were observed

were tested: (I) CIM DEAE and 20 mM MES pH 6.0, and (II) CIM

using a Philips CM 100 transmission electron microscope operat-

QA and 20 mM sodium-acetate pH 5.5.

ing at 80 kV, and images were acquired with an ORIUS SC200 CCD

camera using Digital Micrograph Software (Gatan Inc., Pleasanton,

2.4. Infectivity test

CA, USA)

Chromatographic elution fractions containing TYMV and TBSV

2.8. Chemicals

were used for mechanical inoculation of B. rapa L var. rapa and

N. benthamiana Domin, respectively, without prior salt removal.

The MES (2-(N-Morpholino) ethanesulfonic acid) hydrate,

A small amount of silicon carbide (SiC) was dusted onto two ®

Trizma base (Tris(hydroxymethyl) aminomethane), HEPES (2-

leaves per plant, and the elution fractions were gently rubbed in.

[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid), and SDS

Infectivity was conﬁrmed by observing the development of virus

(sodium dodecyl sulphate) were purchased from Sigma (St. Louis,

characteristic symptoms and by testing the plants with TYMV- and

MO, USA). The sodium acetate trihydrate, sodium dihydrogen phos-

TBSV-speciﬁc RT-qPCR assays.

phate dehydrate, sodium hydrogen phosphate dehydrate, glycerol,

acetone, and Bromophenol Blue were obtained from Kemika

2.5. Sodium dodecyl sulphate-polyacrilamide gel electrophoresis

(Zagreb, Croatia). MOPS (3-(N-morpholino)propanesulfonic acid),

(SDS-PAGE) ®

2-mercaptoethanol, and Coomassie Brilliant Blue were from Fluka

(Buchs, Switzerland). The sodium chloride was from Lach-Ner (Ner-

The protein composition of the chromatographic fractions was

atovice, Czech Republic). The sodium hydroxide was from TTT d.o.o.

analyzed after acetone precipitation by SDS-PAGE and staining with

(Sveta Nedjelja, Croatia). The 40% acrylamide and 2% bis-acrylamide

Coomassie Brilliant Blue solution.

solutions were from Merck (Darmstadt, Germany). The methanol

and acetic acid were from Carlo Erba (Milano, Italy). All the buffers

2.6. Nucleic acid extraction and estimation of recovery for TYMV,

were ﬁltrated through 0.22 ␮L PES membrane ﬁlters from TPP (St.

TBSV, and TMV in chromatographic fractions by RT-qPCR

Louis, MO, USA).

A QIAamp Viral RNA Kit (Qiagen, Valenica, CA, USA) was used to

3. Results

isolate total RNA from the fractions. Two nanograms of luciferase

control RNA (LUC, Promega, Madison, WI, USA) were added per

3.1. Partial puriﬁcation of plant viruses with ion-exchange

sample immediately prior to isolation to account for discrepancies

chromatography on monolithic supports

between samples during the isolation process and/or RT-qPCR.

A high-capacity cDNA Reverse Transcription Kit (Applied Biosys-

Previous chromatographic optimisations had to be accom-

tems, Carlsbad, CA, USA) was used to synthesize cDNA according to

plished for each particular virus to enable the ﬁnal decision on

the manufacturer’s instructions. We also included an initial dena-

◦

conditions necessary to fulﬁl the main task: separation of viruses

turation step at 95 C in a 2720 Thermal Cycler (Applied Biosystems,

® from each other. In the scope of those experiments, we partially

Carlsbad, CA, USA). qPCR reactions were performed with SYBR

puriﬁed three plant viruses directly from plant tissue using the

Premix Ex TaqTM (Tli RNaseH Plus, TaKaRa, Shiga, Japan). The cDNA

chromatography-based approach described in Section 2.2. Prior to

was diluted 10 times to avoid any potential inhibition caused by

chromatography, the samples were homogenized and ﬁltered as

large amounts of DNA or other inhibitors potentially present in the

described in Section 2.1.

sample [47]. Two microliters of the diluted cDNA were applied to

the TYMV-, TBSV-, or TMV-speciﬁc qPCR assays in duplicate, as well

as to the LUC-speciﬁc qPCR assay in 20 ␮L ﬁnal volume. Primer 3.1.1. Turnip yellow mosaic virus

sequences are available in Table S1. The primers’ concentrations In a preliminary experiment, we tested whether TYMV from

and cycling conditions were deﬁned according to the manufac- the TYMV-Y isolate would bind to an anion exchanger at neutral

turer’s instructions. The qPCR was performed on a 7300 Real-Time pH. The resulting chromatogram is available in the Supplement

PCR System (Applied Biosystems, Carlsbad, CA, USA). The Ct was (Fig. S2A). The A260/A280 height ratio for the peak present in elution

72 J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78

fraction E2 was 1.63 ± 0.01. This value is in the range characteris-

tic of plant icosahedral viruses and is very close to the value range

associated with the pure TYMV [26]. In the RT-qPCR analysis of

the elution fractions, viral RNA was detected in elution fractions

E2 and E3. This coincided with the presence of a 20 kDa protein

band that corresponded to the TYMV coat protein (CP, Fig. S2A,

insert). No TYMV RNA and no CP were detected in the ﬂow-through

and elution fraction E1. Elution fraction E2 was clearly enriched in

TYMV; therefore, we used it without prior desalting for the inoc-

ulation of healthy turnips. Infectivity was conﬁrmed by observing

the development of TYMV characteristic symptoms and by testing

the inoculated plants with the TYMV-speciﬁc RT-qPCR assay. When

we compared the chromatographic proﬁle and the SDS-PAGE anal-

ysis of the collected fractions from non-infected turnips (Fig. S2B)

with the results obtained with TYMV-infected homogenate (Fig.

S2A), we found several differences. Firstly, the virus peak present

in the elution fraction E2 seen in the TYMV-infected homogenate

chromatographic proﬁle was absent in the same fraction in the

non-infected homogenate chromatographic proﬁle. Secondly, in

the SDS-PAGE analysis, the TYMV CP represented by 20 kDa pro-

tein band was not present either in the non-infected homogenate

or in the analyzed fractions obtained after chromatography with

the non-infected homogenate. Based on the A260/A280 height ratio

of 0.52 ± 0.00 for the peak present in elution fraction E3 in the

non-infected homogenate chromatographic proﬁle and the SDS-

PAGE analysis of the same fraction, we conclude that this fraction

is composed of plant proteins, and that it is dominated by Rubisco.

TYMV is characterized by pH-dependent stability [29]; there-

fore, to further optimize the chromatographic puriﬁcation of

TYMV-Y, we analyzed recoveries from both anion exchangers under

different buffer pH conditions ranging from pH 5.5 to pH 9.0. The

A260/A280 ratios for the TYMV-Y peaks are available in the Supple-

ment (Table S2). Based on the presence of a TYMV-Y distinctive

peak in chromatograms obtained within pH range 5.5–8.0 (Fig. S3),

we concluded that the virus is stable under these conditions. At pH

9.0, the chromatographic proﬁles on both columns lacked a TYMV-

Y distinctive peak, and the recoveries from the CIM QA column

were extremely low (Fig. 1A). The relatively high recovery from

CIM DEAE at pH 9.0 can be explained by the loss of charge that

the DEAE undergoes at that particular pH. This results in a weaker

interaction between the binding molecules and the column. How-

ever, in this case everything that was bound to the column, TYMV-Y

as well as other parts of the plant tissue homogenate, eluted in

one single peak. These results are in agreement with the literature,

Fig. 1. (A) TYMV recoveries obtained under different buffer pH conditions (pH

which states that, above pH 8.5 TYMV particles begin to disintegrate 5.5–9.0) on CIM DEAE and CIM QA. The chromatographic buffers are listed in Table 1.

[51]. The chromatograms and SDS-PAGE analysis of fractions of TYMV-Y obtained with

buffer pH 6.0 on (B) CIM-DEAE and (C) CIM-QA columns. The samples (300 ␮L) were

Recoveries, ranging from 36% to 95%, were obtained on both

applied with 20 mM MES pH 6.0 and eluted using a linear gradient. The elution

columns within the range of pH 6.0–8.0 (Fig. 1A). The A260/A280

buffer was the same as the loading, with the addition of 2 M NaCl. The ﬂow rate was

ratios (Table S2) conﬁrmed that relatively pure virus fractions could 2 mL/min. Runs were performed in triplicate. M – MW marker, FT – ﬂow-through,

be obtained from CIM DEAE at that pH range. On the CIM QA column, E1–E4 – elution fractions, – indicates the fraction in which the majority of plant

host proteins (mostly Rubisco) were eluted.

the optimal pH range was between pH 7.0 and pH 8.0. It seems that,

at lower pH levels, the CIM QA column did not effectively separate

plant protein contaminants from the virus. The SDS-PAGE analysis

additionally conﬁrmed a better separation potential of CIM DEAE 3 and 4 min that was present only in the TBSV-infected homogenate

in contrast to QA (Fig. 1B and C). Taking into account that CIM DEAE proﬁle (Fig. S4A) with an A260/A280 height ratio of 1.73, which

∼

and pH 6.0 provided the virus fraction which came the closest to the is close to the value reported for the pure virus [26]. A 40 kDa

pure virus A260/A280 value (Table S1) in addition to high recoveries, band representing TBSV CP was abundant in the E2 fraction; there-

we selected these conditions for further experiments. fore, it was used to inoculate healthy N. benthamiana plants. The

inoculated plants showed virus characteristic symptoms and virus

3.1.2. Tomato bushy stunt virus presence was conﬁrmed with the TBSV-speciﬁc RT-qPCR assay.

Since TBSV is stable within a broad pH range [52], we decided Thus, TBSV maintained its infectivity after chromatography on the

to apply the same column and chromatographic buffers that were CIM DEAE column.

optimized for TYMV, with a simple linear gradient for elution. In In order to determine TBSV recovery and particle integrity, we

the initial experiments (Fig. S4), we observed clear differences repeated the chromatographic runs using load prepared from plant

between the chromatographic proﬁles of the infected and unin- tissue infected with elution fraction E2 from Fig. S4A. Again, a dis-

fected homogenates. There was a differential peak located between tinctive peak was observed at the same position between 3 and

J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78 73

eluted later in the gradient. This recovery is similar to recover-

ies obtained for ToMV by semi-quantitative ELISA [17]. Fraction

E5b was clearly enriched in TMV (Fig. 3); therefore, we examined

it under an electron microscope (Fig. 3 insert) and used it in the

following experiments.

3.2. Analysis of TYMV isolates belonging to two strain groups

The Edinburgh isolate (TYMV-E) and the Yugoslavia isolate

(TYMV-Y) both belong to the TYMV-1 group. They are serolog-

ically and biologically identical [46]. TYMV-E is the type isolate

and TYMV-Y is the isolate commonly used in our laboratory. We

decided to investigate whether they also have the same or similar

chromatographic proﬁles. The Northumberland isolate (TYMV-N)

belongs to the TYMV-2 group and can be serologically differentiated

Fig. 2. Chromatogram of N. benthamiana homogenate infected with TBSV fraction

from TYMV-E and TYMV-Y with an Ouchterlony double immunod-

E2 described in Fig. S4A. Insert: electron micrograph of fraction E3. TBSV recoveries

iffusion assay [53] (Fig. S5). We wanted to determine whether, in

are indicated next to the fractions. The loading buffer was 20 mM MES pH 6.0, and

addition to the serological difference, there would be changes in

the elution buffer was the same, with the addition of 2 M NaCl. The sample (100 ␮L)

was loaded onto the CIM DEAE column. The ﬂow rate was 2 mL/min. Runs were the virus retention time.

performed in triplicate. FT – ﬂow-through, E1–E6 – elution fractions. As expected, the virus peaks from the TYMV-1 isolates

overlapped (Fig. 4). The slight differences in the overall chromato-

graphic proﬁles could be attributed to plant tissue variability. The

4 min in the elution fraction E3 (Fig. 2), and the A260/A280 height

TYMV-speciﬁc RT-qPCR assay and the SDS-PAGE analysis also con-

ratio of 1.63 ± 0.02 suggests that the purity of this preparation is

ﬁrmed the location of the virus (Fig. S6). The chromatographic

greater than that provided by the initial experiments. Together with

proﬁle of TYMV-N was signiﬁcantly different, with a shorter reten-

the slight differences between the proﬁles, this could be attributed

tion time than the TYMV-1 viruses (Fig. 4). The location of the

to host-inﬂuenced sample variability. The integrity preservation of

virus was conﬁrmed by SDS-PAGE analysis (Fig. S6C) and electron

the particles was conﬁrmed by analysing fraction E3 by electron

microscopy (Fig. S7), but the TYMV speciﬁc RT-qPCR assay did not

microscopy (Fig. 2 insert). According to the RT-qPCR results, the

provide conclusive results. After comparing the primer sequences

majority of the TBSV with the recovery of 68.9% was in elution

with TYMV coat protein gene sequences available in the NCBI

fraction E3 and 9.5% of TBSV was in elution fraction E4. The lat-

Nucleotide database, we concluded that they are speciﬁc for TYMV-

ter encompassed the tail of the E3 peak and, taken together, these

1 isolates and that they cannot amplify TYMV-2 CP nucleotide

two fractions give a recovery of ∼80%. An additional 11.7% eluted in

sequences. The morphology of the TYMV-E and TYMV-N viruses in

fraction E5, and this result matched the previous SDS-PAGE fraction

elution fractions was also compared by electron microscopy (Fig. 5),

analysis (Fig. S4A), where the CP band was abundant in one frac-

and the particles in both fractions had a similar size (28–30 nm).

tion and small amounts eluted in the following fractions. A small

Although the classical negative staining method is not enough to

TBSV-RNA percentage of 5.4% was found in fraction E6. The peak

determine the shape thoroughly, it seems that TYMV-E and TYMV-

position and A260/A280 ratio of 2.09 ± 0.07 suggests that this fraction

N particles display some distinct differences in particle structure

probably contained free unencapsidated genomic RNA. The recov-

e.g., TYMV-E particles have sharper edges, which was also noticed

ery value matches that of Potato spindle tuber viroid RNA and COX

in the case of incompletely formed particles. The TYMV-E icosahed-

mRNA obtained under the same chromatographic conditions [48].

ral structure is well documented [28,29], but the atomic structure

TBSV efﬁciently binds to an anion exchanger at pH 6.0, and it

of the latter virus is not known. Further research is necessary to

remains infective after elution. High recoveries were obtained, but

fully characterize isolates belonging to the TYMV-2 group.

the purity of the TBSV containing fractions depends on individual

load variability probably inﬂuenced by the post-inoculation time

and season.

3.3. Separation of individual viruses present in a mixed sample

3.1.3. Tobacco mosaic virus Based on the differences in the elution patterns displayed by

Tomato mosaic virus (ToMV), a close relative of TMV, was previ- TYMV-E/TYMV-Y, TYMV-N, TBSV, and TMV, we decided to test the

ously puriﬁed and concentrated with monolithic supports [16,17]. hypothesis that monolith chromatography can separate individual

Thus, for TMV partial puriﬁcation, we followed the conditions viruses present in a mixed sample. Chromatographic fractions con-

described earlier with the following modiﬁcations: the concentra- taining individual viruses from Section 3.2 were used to prepare

tion of NaCl was raised to 2 M and the linear gradient separation artiﬁcially mixed samples. The loading sample volumes were

was implemented. adjusted to yield chromatographic peaks of similar heights. Prior to

There was a clear difference between the chromatographic mixing, chromatographic experiments were performed with indi-

proﬁle of TMV-infected and non-infected N. megalosiphon vidual viruses; that is, the runs were performed with the same

homogenates (Fig. 3). A new peak emerged when infected volumes of the individual viruses that were later used to prepare

homogenate was loaded. The peak in question, located in elu- the mixed samples. Two sets of conditions were tested: (I) 20 mM

tion fraction E5, had an A260/A280 height ratio of 1.15 0.00, a MES pH 6.0 and CIM DEAE, (II) 20 mM sodium-acetate pH 5.5 and

value similar to that of the pure virus [26]. SDS-PAGE revealed a CIM QA. The A260/A280 height ratios obtained from individual runs

17.5 kDa protein band corresponding to TMV CP in all the fractions (Table S3) together with the SDS-PAGE results (data not shown)

obtained from the TMV chromatogram, being most abundant in suggest that, after a second round of chromatography, we achieved

fraction E5. This protein was absent in the fractions from the non- a higher degree of purity with some of the viruses. The model

infected plant chromatogram. Nevertheless, the RT-qPCR results viruses were certainly puriﬁed enough to enable clear recognition

∼ ∼

showed that 74% of the virus eluted in fraction E5, whereas 8% of the respective separated fractions.

74 J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78

Fig. 3. (A) Chromatograms of non-infected and TMV-infected N. megalosiphon homogenates with an electron micrograph of fraction E5b from the TMV chromatogram (insert).

SDS-PAGE analysis of the fractions obtained from (B) non-infected and (C) TMV-infected homogenates. TMV recoveries are indicated above the fractions. The loading buffer

was 20 mM sodium acetate pH 5.5, and the elution buffer was the same, with the addition of 2 M NaCl. The samples (500 ␮L each) were loaded onto the CIM QA column. The

ﬂow rate was 2 mL/min. Runs were performed in triplicate. M – MW marker, FT – ﬂow through, E1–E7 – elution fractions. E6 and E7 were only analyzed by RT-qPCR and not

by SDS-PAGE.

1400 100 TYMV-N 3.3.1. DEAE and pH 6.0

TYMV-2

TYMV-Y 90 First, we decided to test the conditions optimized for TYMV

1200

TYMV-E partial puriﬁcation and successfully applied to TBSV. Overlapping

U) 80 fer) f

chromatograms of runs performed with individual viruses are 1000 gradient bu (m 70

available in the supplement (Fig. S8A). ion

800 Then we mixed the four viruses and separated them into four

TYMV-1 50 individual peaks (Fig. 6). From previous chromatographic exper-

at 260

600 iments (Fig. S8A) and SDS-PAGE results (Fig. 6B), we concluded ce 40

that TBSV eluted in fraction E1 (A260/A280 = 1.65); TYMV-N in E2

rba 30

400

dient (% dient (% of elut (A260/A280 = 1.61); TYMV-E in E3 (A260/A280 = 1.63); and TMV in

20 Abso E4 (A /A = 1.17). The A /A height ratios indicated that Gra 260 280 260 280

200

10 fractions E1–E3 contained icosahedral viruses and E4 contained

a rod-shaped virus. In addition to virus separation, this was the

0 0

01234567891011 ﬁrst time that two virus strains, TYMV-1 and TYMV-2, were sep-

Time (min) arated from the same sample. Furthermore, baseline separation

between the two was achieved. The chromatogram and SDS-PAGE

Fig. 4. Chromatograms of three different TYMV isolates. The loading buffer was

results (Fig. 6A and B) reveal that baseline separation was not

20 mM MES pH 6.0, and the elution buffer was the same, with the addition of 2 M

achieved between fractions E3 and E4. Fractions E3/4 and E4 con-

NaCl. The samples (400 ␮L of TYMV-Y, 500 ␮L of TYMV-E, and 300 ␮L of TYMV-N)

tain a ∼35 kDa protein band that could be attributed to TMV CP

were loaded onto the CIM DEAE column. The ﬂow rate was 2 mL/min. Runs were

performed in duplicate. dimers. Electron micrographs conﬁrmed the presence of icosahed-

ral viruses in E1–E3 and a rod-shaped virus in fraction E4 (Fig. 6C–F).

They also revealed signs of virus particle decay: the majority of

icosahedral viruses were positively and not negatively contrasted,

and the rod-shaped particles seemed to be broken. The reasons

for the observed particle damage after successive runs could be

J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78 75

maize lethal necrosis, cassava mosaic disease, and sweet potato

virus disease [3]. Tomato leaf curl disease, for instance, is caused

by members of the Begomovirus genus when a mixed infection

consisting of two distinct begomovirus species leads to pseu-

dorecombination with direct consequences on the development

of the disease [54]. Different combinations of potyviruses with

other viruses have been described, as well [3]. For example, PVY

and icosahedral Potato leafroll virus (PLRV), two aphid-transmitted

viruses that have the most devastating impact on potato produc-

tion worldwide, often occur in mixed infections, but the nature

of their interaction is not well understood [55,56]. Compared to

plants infected with only one of these viruses, PVY or PLRV, plants

infected with both viruses developed more pronounced symptoms,

which had an impact on the increased fecundity of two virus vec-

tors, Myzus persicae and M. euphorbiae [56]. PVY puriﬁcation by

monolithic support has recently been reported [12], and there-

fore, once the PLRV binding/elution conditions are optimized, an

approach similar to the one described here could be used to rapidly

detect mixed infections and separate these two viruses. In light

of the economic impact, quick detection of mixed virus infection

and the separation of individual viruses for further characteriza-

tion are of great importance. Thus, the aim of this work was to

elucidate whether monolith chromatography with CIM columns

can be utilized for such a purpose.

Since chromatographic separation in ion-exchange mode is

based on different charge properties, it is possible to separate virus

particles of similar structure and morphology as long as they have

different net charge and/or charge distribution on their outer sur-

faces. Different salt concentrations are necessary for their elution,

and the strategy could be applied to any mixed infection as long

as the infecting viruses have different isoelectric points. A sim-

ple separating technique was reported for Cowpea chlorotic mottle

virus (CCMV) and Cucumber mosaic virus (CMV) mixed infection

[57]. The technique in question relies on the fact that these two

Fig. 5. Electron micrograms of (A) TYMV-E elution fraction E3 (from Fig. S6B) and

viruses have different systemic-spreading principles. In cowpea

(B) TYMV-N elution fraction E3 (from Fig. S6C).

plants, CCMV causes a systemic infection and maintains its pres-

ence in the inoculated leaves while, CMV spreads systemically, but

one or more of the following: salt, pressure, shear forces, storage,

leaves the inoculated leaves, which remain virus-free. In tobacco

temperature changes, etc.

plants, only CMV causes systemic infection, while CCMV remains

localized in the inoculated leaves. It is quite easy to separate the

3.3.2. QA and pH 5.5 two viruses by managing them in different experimental hosts.

In the second condition set where we used CIM QA and buffers Although simple, this technique requires weeks of waiting until the

with pH 5.5, individual chromatograms (Fig. S8B) showed that a dif- plants amplify enough of each virus to develop characteristic symp-

ferent quality of separation was achieved. TBSV again eluted ﬁrst, toms. Since the two viruses have different isoelectric points (CCMV

but half of the sample seemed to be in the ﬂow-through. This may pI 3.7, CMV pI 5.5 [26]), it is reasonable to assume that CCMV and

have been caused by salt remaining from the previous chromato- CMV mixed infection could be detected and even separated much

graphic runs and preventing the virus from binding to the column. faster with the approach described in this paper.

Another possible reason could be that the pH value of the work- Furthermore, we believe that ion-exchange chromatography on

ing buffer was not appropriate. Better baseline separation between monolithic supports could be used as a preliminary analysis tool in

the two TYMV strains was achieved, but the TYMV-E was barely investigations of various virus strains. Several different adenovirus

separated from the TMV. According to the A260/A280 ratios, all the serotypes and two adenovirus chimeras have been previously ana-

viruses except TBSV achieved higher purity (Table S3). Neverthe- lyzed by anion exchange chromatography using particle-based

less, the locations of the icosahedral particle structure/morphology columns [58,59]. The obtained retention times were serotype spe-

were recognized on the chromatographic proﬁle of the mixed sam- ciﬁc [58] and it was also shown in experiments with adenovirus

ple (Fig. 7). The A260/A280 ratios for E2 where TYMV-N eluted chimeras that, in the case of anion-exchange chromatography, the

and E3 where TYMV-E eluted were 1.53 and 1.54, respectively. The adenovirus retention time was determined by the composition of

A260/A280 ratio for E4 where TMV eluted was 1.2. the hexon protein [59], which is the most abundant capsid pro-

tein in adenovirus particle. By comparing the chromatographic

4. Discussion behaviour of several TYMV isolates, we found different retention

times for isolates that belong to serologically distant strain groups

Today, there is increasing evidence that mixed virus infections TYMV-1 and TYMV-2. As reported for TYMV [60], it is possible

are common in nature and not simply rare occurrences. The compo- for one plant to be infected with more than one strain of the

sition of virus species present in the mix can inﬂuence the disease same virus. By separating TYMV-N and TYMV-E from the artiﬁcially

onset and symptoms development, and we are only beginning to mixed sample, we showed the potential of chromatography for the

understand various mechanisms of virus–virus interaction [3,4]. detection/monitoring of virus strains in a certain host. Such appli-

Examples of devastating mixed infections of plant viruses include cation could be extended to other virus hosts and/or systems. For

76 J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78

Fig. 6. (A) Chromatogram of a mixed sample (in total 140 ␮L = TBSV 38 ␮L, TYMV-N 10 ␮L, TYMV-E 32 ␮L, TMV 60 ␮L) analyzed with the CIM DEAE column and (B) SDS-PAGE

analysis of the fractions. The loading buffer was 20 mM MES pH 6.0, and the elution buffer was the same, with the addition of 2 M NaCl. The ﬂow rate was 2 mL/min. Runs

were performed in duplicate. Electron micrographs of elution fractions: (C) E1, (D) E2, (E) E3 and (F) E4. TBSV – Tomato bushy stunt virus, TYMV-N – Turnip yellow mosaic virus

Northumberland isolate, TYMV-E – Turnip yellow mosaic virus Edinburgh isolate, TMV – Tobacco mosaic virus, M – MW marker, L – load, FT – ﬂow-through, E1–E4 – elution

fractions.

example, it could be of great help in the research of the emerging chromatographic separation in the scope of monitoring and under-

Dengue virus (DENV), whose serotypes cause different symp- standing the particular impact of each one.

toms and inﬂuence disease development [61]. It is estimated that When using PCR and serological methods (e.g. ELISA) for the

each year there are 360 million DENV infections worldwide, out generic detection of viruses, non-targeted ones can be overlooked

of which approximately 96 million cases develop symptoms of due to the lack of speciﬁc primers or antibodies. Next-generation

various severity [62]. There are four DENV serotypes that are co- sequencing is the best tool for detection and new virus discovery,

circulating in different regions, and the discovery of a ﬁfth serotype but due to the cost, complex sample preparation, and data analy-

has recently been reported [63]. DENV infection provides lifelong sis, it is still not accessible to all researchers. Another disadvantage

immunity to a particular serotype, but does not provide cross- is that the method is time-consuming and does not provide pure

protection against others. This information suggests signiﬁcant infective viruses, which are necessary for further virus characteri-

differences among serotypes, and hence the feasibility of their zation. The strategy used here, i.e., CIM monolithic chromatography

J. Rusˇciˇ ´c et al. / J. Chromatogr. A 1388 (2015) 69–78 77

separation on CIM QA using 20 mM sodium acetate pH 5.5. Mono-

lith chromatography could be applied in the preliminary detection

of mixed virus infections, as well as in the separation of viruses

from mixed samples.

Acknowledgments

This work was supported by grant number 119-1191192-1222

of the Croatian Ministry of Science, Education and Sports and by the

Slovenian Research Agency through program P4-0165 and project

L4-5525. The authors would like to thank Dr. Thomas Sheppard and

Dr. Alexander Hoyt for their critical reading of the manuscript and

–

Dr. Ðorde Mamula for providing background information on the

TYMV isolates.

Appendix A. Supplementary data

Fig. 7. Chromatogram of a mixed sample (in total 200 ␮L = TBSV 80 ␮L, TYMV-N

40 ␮L, TYMV-E 40 ␮L, TMV 40 ␮L) analyzed with the CIM QA column with SDS-PAGE

analysis of the fractions (insert). The loading buffer was 20 mM sodium acetate pH Supplementary data associated with this article can be

5.5, and the elution buffer was the same, with the addition of 2 M NaCl. The ﬂow found, in the online version, at http://dx.doi.org/10.1016/j.chroma.

rate was 2 mL/min. Runs were performed in duplicate. TBSV – Tomato bushy stunt 2015.01.097.

virus, TYMV-N – Turnip yellow mosaic virus Northumberland isolate, TYMV-E – Turnip

yellow mosaic virus Edinburgh isolate, TMV – Tobacco mosaic virus, M – MW marker,

L – load, FT – ﬂow-through, E1–E4 – elution fractions. References

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