Separation of adenovirus by chromatography on DEAE-cellulose

264

DISCUSSION

TABLE

AND

PRELIMIKARY

1

EFFECT OF TMV AND VIRUS Y ON THE NUMBER OF LESIONS PRODUCED BY VIR~JS X Virus X lesions* in experiments:

i

Inocula and infectivity ratiosa

lc -_

A. “Type” TMV + virus X B. Virus X alone

2

3 __

4

5

705lfi6817243485826

369 114 4061653425 Lesions from A/ , 1.914.6 4.2 2.11.9 Lesions from B --_-----_---

6

7

871554 568 96 1.55.8

a Inocula of TMV and virus Y were infective 6 TMV was purisaps except that in experiment fied by six cycles of high and low speed centrifugation and contained 0.5 mg. TMV per milliliter distilled water and in experiment 7 TMV was purified by four precipitations with ammonium sulfate and contained 1.4 mg. TMV per milliliter. Virus X inoculum was infective sap diluted lO-3 in distilled water. b Total number of lesions on 12 half-leaves of

3~‘icotiana tahacum L. (var. White Burley). c In experiments 1-4 virus X was inoculated 6 days after TMV or virus Y. In experiments 67 equal volumes TMV or virus Y and virus X were inoculated simultaneously. Differences between A and B significant at P = 0.01.

2

EFFECT OF LEAF AGE ON ABILITY OF VIRGS Y TO INCREASE THE NUMBER OF LESIONS PRODUCED BY VIRUS X Infectivity

ratiosb on leaves

Experiment”

1 2 3 4

hypothesis comes from the fact that virus X lesions are usually more clearly defined in the presence of TMV or virus Y. The present phenomenon may be useful for increasing the sensitivity with virus X.

A. Virus Y + vi- 1803 820118916996203246360 rus X B. Virus X alone 48 53 437 36128 57172 Lesions from A/ ‘9.2115.51 2.7147.214.856.912.1 Lesions from B

TABLE

REPORTS

Young

Medium

Old

3.2 29.0 2.2 8.5

1.4 12.0 2.0 3.0

1.0 4.3 1.1 2.2

a Equal volumes virus Y + virus X and Virus X alone inoculated to 4 half-leaves of each age group. Lesions on tobacco leaves inoculated with virus Y + virus X b Lesions on tobacco leaves inoculated with virus X

of infectivity

tests

REFERENCES 1. BEXNETT, C. W., Advnnces in Virus Research 1, 39-67 ( 1953 ) 2. ROCHOW, W. F., and Ross, A. F., Birologv 1, IO-27 (1955). S. TIIO~OK, A. D., LVature 181, 1547-1548 (1958). A. D. THOMSON Plnnt Diseases Division Department of Scientific and Industrial Research Christchurch, New Zealand Received October Y, 1960

Separation

of Adenovirus

by Chromatography

on DEAE-Cellulose

This report is chiefly concerned with the results of the purification and the chromatographic differences among three types of adenovirus, belonging to different subunits (1). Prototype strains of adznovirus types 1 (Ad. 71), 3 (G. B.) and 8 (Trim), propagated on HeLa cell cultures, were used in these experiments, The growth medium consisted of 0.5% lactalbumin hydrolysate (N.B.C.), 0.1% yeast extract (Difco) in Hanks’ solution supplemented with 20% calf serum. DEAE-cellulose type 20 (Brown Co., U.S.A.) was used in columns (1 x 10 cm) treated with 0.005 M borate buffer at pH 8.1 and loaded with 15-50 ml of the infected HeLa culture fluid, previously dialyzed against distilled water. The gradient elution method was used in all experiments. A continuously increasing concentration of NaCl in 0.005 d4 borate buffer at pH 8.1 was run onto the column and 25 eluates were collected in 3-ml amounts. The final NaCl concentration was 0.5 M. Protein was measured spectrophotometritally at 750 rnp after the Folin-phenol reaction (Z), and nucleic acid was estimated spectrophotometrically at 260 and 280 mp. In order to measure infectivity, aliquots were taken from each fraction, centrifuged at 8000 rpm for 30 minutes at 4”C, and the

DISCUSSION

AND

PRELIMINARY

supernatants obtained were tested in duplicate in HeLa cell culture tubes. The tubes were incubated in a roller drum at 37” and observed daily for a week for cytopathic effect. The fractions which showed the cytopathic effect of adenovirus were retested for infectivity by diluting with Hanks’ balanced salt solution in log 4” steps, and adding 0.1 ml of each dilution to HeLa cell tubes. The end points were read by CPE after 7 days’ incubation.

In some experiments an equal volume of antiserum of appropriate dilution was added to each fraction. The virus-antiserum mixture was allowed to interact for 1 hour at 37”, and then O.l-ml aliquots were added to HeLa tissue culture tubes in duplicate. Neutralization tests were scored 48 hours after the first evidence of definite cytopathic effect appeared in the control tubes. Early cytopathic effect (ECE) (5-5) due to toxinlike material was observed 24 hours after the inoculation of each eluate.

4..

0 _

Type- I

3.. 2..

% g

-/--

__-I .. *_-- -0-c r 4--

TCID,o ECE - -_--

__--

_- --

--0.5 -0.4 .:23 .“O.l

r

Type-3

3--

12 5: I20

265

REPORTS

E

__--

_---- __-- _---

0

5

.g E z

IO Fraction

15

20

_/--

-Eiz

E 5b

-81 -0. I

H

25

number

0.9 r

0.8 .. 0.7 ” 0.6

Fraction

number

FIQ. 1. Infectivity and optical density of the eluates of adenovirus type 1,3, and 8 infected tissue culture fluid chromatographed. in a DEAE-column. 0-O Denotes optical density at wavelength 260 rnp; O-O denotes optical density at wavelength 280 mp; X-X denotes optical density at wavelength 750 rnp ; - - - - denotes molar concentration of Nacl.

266

DISCUSSIOX

AND

PRELIMINARY

The infectivity of adenovirus type 3 was eluted at approximately 0.3 M NaCl (tubes no. l&17), whereas nonviral protein in the maintenance medium was eluted at 0.1-0.3 M NaCl (tubes no. 6-14), as shown in Fig. 1. About 80% of the infectivity of the starting material was recovered. Adenovirus type 8 was eluted at 0.25 iVl NaCl (tubes no. 11-B), which was slightly lower salt concentration as compared with that for adenovirus type 3. The recovery of infectivity was almost lOO%, the best among the three virus types used in this experiment. Adenovirus type 1 was eluted between 0.4 M and 0.5 M NaCl, as shown in Fig. 1; it was remarkably different from types 3 and 8 described above. The recovery of infectivity was about 60%. Also, so-called toxinlike material was separated from infective virus and was eluted at approximately 0.2 M NaCl. Tubes no. 9, 10, and 11 showed early cytopathic effect 18-24 hours after inoculation of undiluted eluates. Since it was found that the three types of adenovirus were eluted at different salt concentrations from DEAE-cellulose col-

REPORTS

umns, we attempted to see whether a mixture of types 1 and 3 could be separated by DEAE-cellulose column chromatography. Equal amounts of HeLa cell culture fluids infected with types 1 or 3 were mixed, loaded on the DEAE-cellulose column, and fractionated as in the foregoing experiments. Each of the eluates was analyzed chemically and its infectivity assayed. As can be seen in Fig. 2, each virus type was separated and eluted at the NaCl concentration characteristic for that type. Furthermore, cross neutralization experiments with antisera against types 1 and 3 showed a clear-cut separation of the two types of virus. We tested several kinds of cellulose such as DEAE-, ECTEOLA-, and P-cellulose, and found DEAE-cellulose the most suitable for adenovirus. Klemperer and Pereira (6) and Philipson (7) independently reported the same kind of experiments with adenovirus types 2 and 5, but their experimental condition differed from ours. They used 0.01 M phosphate buffer at pH 6.5 and 0.02 M phosphate buffer at pH 7.2, respectively, as the buffer

Type 3

We

1

number Antitype

I

immune Antitype 3 immune Mixture of ontitypes immune

serum serum land 3 sera

tt++ - - - - - - -

----_ ++++t -----

FIG. 2. Infectivity of the fractions obtained through DEAE-cellulose chromatography of the mixture of adenovirus types 1 and 3 tissue culture fluids and their serological identification by neutralization tests using type-specific antisera. - - - - Denotes molar concentration of NaCl. Cytopathic effect after neutralization by anti-immune serum: + positive, - negative.

DISCUSSION AND PRELIMINARY REPORTS

system. Their recovery of infectivity was low and the peak of the activity was diffuse. In our case, the recovery of the infectivity seems more efficient. This difference in recovery may be due to the different assay methods used; the cytopathic effect was read after 7 days’ incubation in the present experiment instead of 20-24 days which they used. Furthermore, in one case, optical densities at 260 rnp of starting material and the eluted virus fraction (type 1: tubes no. 20-24) were 6.0 and 0.02, respectively, and the recovery of infectivity was 60%. Therefore, the specific activity of virus per optical density at 260 rnp was raised up to 180 times that of the starting material. Extracellular virus was used throughout these experiments, and the fractionation of intracellular virus is now in progress. REFERENCES

1. BOYER,G. S., LE~CHTENBERGER, C., and GINSBERG, H. S., J. Ezptl. Med. 105, 195-216 (1957). 2. LOWRY,0. H., ROSEBROUGH, N. J., FARR,A. L., and RANDALL,R. J., J. Biol. Chem. 193, 265275 (1951). 3. PEREIRA,H. G., and KELLY, B., J. Gen. Microbial.

17,517-524 (1957). /t. PEREIRA,H. G., Virology 6,601-611 (1958). 5. EVERETT,S. F., and GINSBERG,H. S., Virology

6,

770-771 ( 1958). G. KLEMPERER,H. G., and PEREIRA,H. G., Virology 9,536545 (1959). 7. PHILIPSON,L., Virology 10,459-465 (1960).

Institute for Virus Research Kyoto University Kyoto, Japan Received SeDtember 22,1%0 Procedure

for Rapid of Phage

267

was fast, efficient, and economical; the concentrated phage showed no loss of biological activity. Small volumes to. be concentrated are put in narrow-diameter dialysis tubing (Visking S/32 or 18/32) which has previously been boiled 10 minutes in water with a trace of Versene added, then washed. The bags are surrounded by dry Carbowax 4000, a high molecular weight polyethylene (available from the Union Carbide

glycol Chemi-

cal Company). Per 100 ml of lysate, 25-30 g of Carbowax is satisfactory, although the speed of concentration may be increased by the use of more Carbowax and smallerdiameter dialysis tubing. Samples of 100 ml may be concentrated at least tenfold within l-2 hours in the cold. Phages h, +X174, and aR showed no loss of biological activity after concentration, the recovery approaching 100%.

Carbowax two

higher

4000 has been compared with molecular

weight

polyethylene

glycols, Carbowax 6000 and Carbowax 20M, as well as a high molecular weight polyethylene oxide, Polyox WSR-35, with regard to concentrating ability. Table 1 shows the recoveries of four lo-ml samples of phage A, 4.5 x 10” per milliliter, after concentration by the above procedure. Carbo-

wax 4000 was the most efficient and fastest of the polymers tested. Carbowax 20M and Polyox were difficult to handle ; upon im-

bibition of liquid from the dialysis bag, ICHIRO HARUNA they became sticky and gumlike. HIDEAKI YAOI REISAKU KONO For large volumes, the lysate is placed in ITARU WATANABE wide-diameter dialysis tubing, open at one end, after being cleared of bacterial debris

Concentration Lysate

A concentration procedure by osmotically forced dialysis, described by McClendon and Somers (1)) has recently been adapted and applied to phage lysates; success with it has prompted associates to try it on other biological materials. For both large and small volumes of phage lysates, in either nutrient or synthetic media, this process

TABLE

1

CONCENTRATING ABILITIES OF POLYETHYLENE GLYCOLS AND POLYETHYLENE 0x11)~ BY OSMOTICALLY FORCED DIALYSIS Polymer

Molecular weight range

Recovery Phage/ml

ml yo --

3000-3700 6.5 X 10'2.0.7100 Carbowax 4000 6000-7500 5.0 X 1Ol2 0.9100 Carbowax 6000 Carbowax 20111 15,000-20,000 2.0 X 1Ol2 2.1 93 >100,000 1.4 X lOI 3.2 99 Polyox WSR-35