Bovine viral diarrhea virus: Affinity chromatography on Crotalaria juncea lectin

Journal of VirologicalMethods, @ Elsevier/North-Holland

2 (1981)

Biomedical

BOVINE VIRAL DIARRHEA

VIRUS: AFFINITY

CHROMATOGRAPHY

ON

JUNCEA LECTIN

CROTALARIA

J. MORENO-LOPEZ' ‘Departments Biomedical

293

293-300

Press

,T. KRISTIANSEN’

of Virology, Faculty

and P. KARSNAS’

of Veterinary

Medicine

Center, Box 585; and ’ Institute of Biochemistry,

and The National

Veterinary Institute,

Uppsala University, Biomedical Center,

Box 756, S- 751 23 Uppsala, Sweden (Accepted

6 January

Attempts lectin

were made

to purify

bovine

viral diarrhea

to Sepharose 2B. A recovery

coupled

tained.

19811

Electron

microscopy

revealed

differing

in sire. Immunodiffusion

between

the desorbed

mostly

of about

de-enveloped

tests with immune

virus and extracts

from infected

on Crotalarib juncea

virus by chromatography 65% of viral infectivity particles,

rather

calf sera showed

after desorption uniform

precipitation

was ob-

in appearance

but

lines of identity

cell cultures.

INTRODUCTION

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Togaviridae (Porterfield et al., 1978). The envelope of BVDV is acquired by budding of cell membranes. When exposed to various procedures of purification, the particles of BVDV readily lose their envelope and their infectivity is lost. Affinity chromatography on immobolised lectins is both an efficient and a gentle

purification method for enveloped viruses (Hayman et al., 1973; Ponce de Leon et al., 1973; Stewart et al., 1973; Kristiansen, 1975; Hoglund et al., 1977). A lectin isolated from sunn hemp seeds, Crotahia juncea (Ersson et al., 1973; Ersson, 1977) was successfully applied to isolate subunits of parainfluenza3 virus (Hoglund et al., 1977). In the present paper, the adsorption of BVDV to C. juncea lectin followed by desorption with 0.2 M lactose was used as a step in purification attempts. MATERIALS

AND METHODS

Virus and cells The highly cytopathogenic and plaque-producing BVDV strain Ug-59 (Borgen, 1963) was propagated in secondary cultures of calf testicle cells. The maintenance medium was Eagle’s MEM with or without 2% horse serum. The virus was harvested on postinoculation day 5 or 6 when the monolayer was completely destroyed. The virus titers

294

only rarely

exceeded

lo6

p.f.u./ml

Plaque titrations

were carried out on monolayers

in Petri dishes under agar overlay following virus adsorption

for 1 h at 37°C.

Virus concentration

The infected cell culture fluid was frozen and thawed three times. The cell debris was removed by centrifugation at 10,000 g for 30 min. To the supernatant fluid, polyethyleneglycol (PEG-6000, Kebo AB, Stockholm, Sweden) was added to a final concentration of 7% followed by addition of 2.2 g of NaCl (to give a final concentration of 0.5 M NaCl). The mixture was allowed to stand overnight at 4°C with constant magnetic stirring and was then centrifuged for 1 h at 1800 g. The pellet was resuspended in l/20 of the initial volume in TEN buffer (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCl) and kept at 4°C overnight. The resulting suspension was clarified by centrifugation at 1000 g for 10 min or pre-purified by gel filtration on Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). The resulting material was stored at -70°C until required. Preparation of’lectin agarose

The lectin of C. juncea was a gift from B. Ersson, who purified it according to his method (Ersson et al., 1973 ; Ersson, 1977)*. 300 ml sedimented Sepharose 2B (Pharmacia Fine Chemicals, Uppsala, Sweden) was suspended in 300 ml distilled water containing 12 g cyanogen bromide (CNBr). The gel was activated by continuous addition of 2 M NaOH to maintain a constant pH of 1 I .6 for 6 min. After washing with ice-cold 0.5 M NaHCOa , pH 8.5, the activated gel was suspended in 300 ml 0.5 M NaHC03 containing 1.2 g C. juncea lectin and the suspension gently rocked overnight at room temperature (22’C). The difference in UV absorbancy at 380 nm before and after coupling indicated that 2 mg per ml sedimented gel was coupled under these conditions. Affinity chromatography

The lectin agarose was suspended

in PBS and packed in a column with a diameter of

5 cm and a height of 6.7 cm, giving a total volume of 130 ml. After washing with PBS with (10 column volumes), 75 ml of virus-containing sample was applied at a flow rate of 100 ml/h, equalling 5 ml/cm’/h. The virus-containing sample was obtained by concentrating the infected culture fluid 20 times from 1500 ml by the use of PEG-6000 as described above. After passage of the sample, the column was washed with 1.3 I PBS at the same flow rate and then eluted with 0.2 M lactose in PBS. The flow rate was again 100 ml/h and 10 ml fractions were collected. In control experiments, a similarly prepared sample of uninfected culture fluid was chromatographed under identical conditions.

* Samples

of this lectin can be requested

from the authors.

295

Fig. 1 shows elution profiles for desorbed BVDV and for material applied sample was uninfected cell culture fluid.

eluted

when the

Electron microscopy One drop of the eluted fractions, obtained as described above, was placed on a Formvar carbon-coated copper grid and negatively stained with 3% ammonium molybdate or 1.5% phosphotungstic acid (PTA). Grids were examined in a Philips EM 300 electron microscope at a magnification from X25,000 to X68,000 at an acceleration voltage of 60 kV.

An antiserum to the eluted BVDV was prepared in calves devoid of antibodies to this virus. Five consecutive subcutaneous inoculations were given at intervals of 3--4 weeks, each inoculation with 5 ml of a virus suspension containing 4.5 X IO6 p.f.u./ml. The suspension was emulsified in Freund’s incomplete adjuvant (Difco Laboratories, Detroit, MI). The calves were bled one week after the last inoculation. An additional calf antiserum to BVDV (‘Danish antiserum’) was a gift from Dr. A. Meyling, Copenhagen, Denmark).

A280 .18

.06

LO

80

120

Elution

Fig. 1. Virus area represents culture

fluid.

desorption material

ILO 200

210 ml

volume

from

a lectin-agarose

desorbed

in control

column experiment

after when

addition

of 0.2 M lactose.

the applied

The shaded

sample was uninfected

cell

296

Immunodiffusion

tests

The original method cation (1957)

of double diffusion in Petri dishes as well as Wadsworth’s modifi-

were used. To prepare

the gel, Special Agar-Noble

was dissolved in TEN buffer as described by Hopkinson

(Difco Laboratories)

et al. (1979).

RESULTS

Recovery

of virus infectivity

Several experiments of BVDV concentration and affinity chromatography on C. juncea lectin were carried out using the procedure as described in Materials and Methods. Data from a typical experiment are presented in Table 1. About 75% of virus infectivity was recovered after concentration with PEG-6000. When a concentrate with a total infectivity of 7.5 X lo8 p.f.u. in 75 ml fluid was chromatographed on the lectin-Sepharose column, about 65% of virus infectivity was recovered. Electron

microscopy

of eluted virus

Fig. 2 shows particles from the peak fraction (Fig. 1) after desorption from C. juncea lectin. The particles differ somewhat in size, but are of a rather uniform appearance. Most of the particles have a smooth surface resembling that of bald virus particles without envelope. In those particles having an envelope, no surface projections are seen. Identification

by immunodiffusion

In order to identify

the eluted

BVDV, this and a crude extract from cells infected

with BVDV were tested against calf antisera to BVDV by double immunodiffusion. TABLE

1

Infectivity

titers of bovine viral diarrhea

cell culture

virus after concentration

and affinity

chromatography

Volume

Infectivity

(ml)

p.f.u./ml

Total p.f.u.

fluid

1500

7

lo9

fluid

75

Material

Infected

Two

x lo5

Recovery

(%)

after clarification Infected

cell culture

after concentration Eluted

pool of fractions

adsorption

7.5 x lo*

4.5 x 106

4.9 x loa

100 (75)a

after

110

65

to C. juncea lectin

The virus infectivity real recovery

lo7

by PEG

was 75%).

recovered

after PEG-6000

concentration

was arbitrarily

considered

100% (the

297

Fig. 2. Electron microscopy of BVDV after desorption from Crotalaria juncea lectin and stained with PTA. The particles with simiku appearance are differing in size. Bar = 200 nm.

or three precipitation lines of identity appeared when either our own or a Danish antiserum to BVDV was applied (Fig. 3). There were no precipitation lines between these antisera and a crude extract from non-infected

cells (not shown).

DISCUSSION

The use of immobilised lectin for the purification of whole virions or viral subunits with exposed carbohydrate chains is an attractive alternative to centrifugation and ultrafiltration techniques, particularly for fragile virus particles that tend to disintegrate even under moderate mechanical stress. The lectin from C. jttnceu is a glycoprotein containing 10% (w/w) carbohydrate. As characterized by Ersson (1977) it consists of four identical subunits, each with a moiecular weight of 3 1,400. Although it is not inhibited by a a-o-glucose, the presence of a

298

Fig. 3. Immunod~u~on tests showing precipitation lines of identity between a calf antiserum to BVDV (upper well), BVDV after adsorption and desorption (lower well, right), and an extract from infected cell cultures (lower well, left).

glucosyl residue adjacent to galactose, as in lactose, melibiose, and raffinose, leads to an increase in affinity. For example, lactose is eight times more efficient in inhibiting hemagglutination than is galactose alone. The lectin does not bind to agarose, indicating that it recognises terminal galactosyl residues rather than internal ones (Ersson et al., 1973). The much higher affinity of the lectin for lactose than for galactose is a useful property in affinity chromatography of viruses and viral glycoproteins, since complete desorption of galactosyl residues is obtained at lactose concentrations as low as 0.1-0.2 M in a narrow elution zone. In addition to complete virions and virus enveiope components, the lectin will bind non-viral material of cellular origin with an appropriate carbohydrate composition. Since cell membrane carbohydrates are incorporated into the peplomers during virus synthesis, the culture fluid will always contain some cellular debris carrying the same carbohydrate determinants as those involved in the adsorption of virions to the lectin (Fig. 1). The extent of this sharing of determinants

is being studied

as a part of the isolation and characterization of the envelope components now in progress. Bovine viral diarrhea virus (BVDV) is notoriously difficult to purify with a high recovery of infectivity. Except for Stott et al. (1974) who reported 90% recovery after purification on a sucrose gradient, authors who used various gradients reported recoveries varying between 17% and 50%, as for instance Maess and Reczko (1968), Parks et al. (1972), and Laude (1979). Utilising C, junceu lectin for adsorption and lactose for desorption of BVDV concentrated by PEG-6000, a recovery of about 65% infectivity was obtained (Table 1). The lectin from Viciu ervilia, successfully used to purify whole influenza virus (Kristiansen, 1975), did not work equally well with BVDV (unpublished). The recovery of virus infectivity and the sharp peak formed by the infectious virus after desorption from C. juncea lectin (Fig. I) shows that chromatography on this lectin

299

might

be a gentle method

for purification

of BVDV. However, only a few enveloped

particles were visualized by EM (Fig. 2), which is in contrast to the recovery of infectivity measured by plaque assays. It is probable that the particles were damaged by the procedure of negative staining. Pre-fixation of the particles with formaldehyde did not improve the results. However, there is also an early suggestion (Friedman and Pastan, 1969) that in togaviruses (Semliki Forest virus) the phospholipid bilayer of the envelope is not necessarily required for infectivity. It might be argued that we did not try to calculate the degree of purification. However, we tried it by labelling the virus during replication with [35 Slmethionine (not shown). But like Felmingham and Brown (1977), we found this method highly inefficient, probably because of the low total yield of virus particles. Besides, these authors complain that ‘in spite of a relative purification of greater than a thousand-fold, it was found that residual host cell proteins were contaminating the virus preparation’. The two or three precipitation lines in immunodiffusion tests (Fig. 3) are due to virus subunits (Matthaeus, 1979) and the soluble antigen (Gutekunst and Malmquist, 1965). Three lines were regularly produced when concentrated extracts of BVDV-infected cells were tested against the Danish antiserum used in this study (Diderholm et al., 1974). ACKNOWLEDGEMENTS

We are indebted to Professor Z. Dinter for helpful advice and critisism, and Mrs. S. Martelius and E. Cafaro for their technical assistance. This investigation was supported by the Swedish Farmers Meat Marketing Association and The Swedish Board For Technical Development.

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