DISCUSSION AND PRELIMINARY REPORTS 221

ACKNOWLEDGMENTS

The authors are indebted to Mrs. G. Lange for capable technical assistance. The work was sup- ported in part by the Deutsche Forschungsge- meinschaft and by the Verein zur Forderung der Erforschung und Beklmpfung der spinalen Kinder- llihmung, Bielefeld.

REFERENCES

1. MANDELL, J. ID., and HERSHEY, A. D., Anal. Bio- them. 1, f&77, 1960.

2. COCITO, C., PRIXZIE, A., DE SOMER, P., Nature 191,573-!575,1961.

3. KUBINSKI, H., KOCH, G., DREES, O., Biochim. et Biophys. Acta in press.

H. KUBINSKI G. KOCH

Laboratorium der Stiftung zur Erforschung der spinalen Kinderlahmwng und der Multiplen Sklerose

Hamburg 20, Germany Received Jan,uary 18,1962

Nomenclature of Enteroviruses

The American Enterovirus committee is suggesting (1) that enteroviruses, includ- ing the Polio, Coxsackie, Echo- and Rhino- (Common cold) viruses should be included in a single numerical series. The committee kindly invited me to attend one of their meetings at which this suggestion was dis- cussed. It was agreed, as a result of my representations, that no formal proposals should be made until the matter had been considered at an international level. This will be done when the International nomen- clature committee and its virus subcommit- tee meet in Montreal in August, 1962.

Some more general suggestions concern- ing virus classification and nomenclature were put forward recently (6) in the hope that in the light of further work and dis- cussion, the international bodies would be better able to make considered proposals in August.

The enterovirus committee’s numerical scheme is quite rightly put forward as a suggestion, and one hopes it will be freely discussed. There might, however, be great

confusion if either of the suggestions I have referred to should lead to premature use cf new names or numbering systems before they have been considered by the appropri- ate international body.

REFERENCES

2. COMMITTEE ON ENTEROVIRUSES, Virology 16, 501- 504 (1962).

2. ASDREWES, C. H., BURTXET, F. M., ENDERS, J. F., GARD, S., HIRST, G. K., KAPLAN, M. M., and ZHDANOV, V. M., Virology 15, 52-55 (1961).

C. H. ANDREWES Chairman, Virus sub-committee, International Nomenclature Committee

Received March 24,1962

An Agar Polysaccharide and d Marker

of Poliovirus

Vogt et al. (1) found that some polio- virus variants (d or d-) form many fewer plaques under an “acid” agar overlay than under a ‘ibasic” one. There was practically no such difference for other variants (d+). Both d and d+ variants multiplied at the same rate, however, in fluid medium, even when the concentration of the bicarbonate corresponded to that in the “acid” overlay. In an attempt to elucidate the role of agar in demonstrating the d marker, we used the observation of Takemori and Nomura (2) that an agar extract inhibited the multipli- cation of the ‘rn mutant of poliovirus pro- ducing minute plaques. We succeeded in showing that this agar extract inhibited the cytopathic action of a d variant in the fluid medium provided this medium contained a low concentration of bicarbonate (3). The agar extract exerted no effect on the cyto- pathic action of a d+ variant. In a continua- tion of this study we have found that by special treatment the agar could be freed from the bulk of the active factor, which turned out to be a sulfated polysaccharide (SPS). The SPS-free agar lost its capacity to inhibit, plaque formation by d variants in the “acid” medium. Accordingly, t’he d character of poliovirus could not be re- vealed by the use of this purified agar. But in the system containing the washed agar

222 DISCUSSIOS AND PRELIMINARY REPORTS

TABLE 1

THE ROLE OF SPS IN THE DEMONSTRATION

OF THE d CHARACTER OF POLIOVIRUS -

2 s 4 - 1

2

-

Concentration of bicarbon- ate in the agar overlay”

Agar / 0.3%

Numbe of

plaques

Bacto-agar 42 Washed agar 46 Washed agar 47

+ SPSh

Bacto-agar 48 Washed agar 42 Washed agar 43

+ SPS6

Size of Jlaque: (mm)

5 5.5-6 5-5.5

6 6-7 6-7

h

P

-

rumbe Size of of llaques

laques (mm)

0 40

0

0 30

0

3-3.5 -

4-5

a Each figure is an average per 3-4 bottjles. The counts were made on the fourth day after inocula- tion with the LSc-Pab strain.

* The final concentration of SPS in the overlay is 1.5 mg/ml.

and added SPS, d and d+ variants could be clearly differentiated.

For purification of the agar and for isola- tion of SPS, 5 g of Bacto-agar (Difco) was suspended in 100 ml of 0.9% NaCl and shaken overnight at room temperature. The supernatant obtained after centrifugation was stored for SPS isolation, and the sedi- ment was treated similarly again. The sedi- ment was then washed with distilled water until the electroconductivity of the wash water only slightly exceeded that of distilled water. The washed agar was dried at 37” and used for experiments. This preparation contained only traces of sulfate as deter- mined by BaS04 formation. BaClz was added to the solution obtained by boiling the agar in 0.5 N HCl for 20 hours (before the addition of BaCl, the solution was di- luted 1: 10). The SPS was precipitated from the supernatants of the agar extracts by the addition of 3 volumes of ethanol at room temperat,ure. After centrifugation the sedi- ment was washed with ethanol and dried at 37”. The preparation gave a positive reac- tion with the Molisch reagent, had no reduc-

ing capacity, and contained 15-17% of sulfate. After hydrolysis in 2% HCl for 3 hours, the preparation became strongly re- ducing. On the basis of these observations, the preparation was considered to be a sul- fated po1ysaccharide.l

The results obtained with the washed agar with and without SPS are presented in Table 1. The methods used for the study of t.he d character were published earlier (6, 7). The results show the inhibition of multi- plication of a d variant under the “acid” overlay to be due to the presence of SPS in the agar. It should be emphasized that at a sufficiently high concentration of bicarbo- nate, or rather at sufficiently high ionic strength and pH (7, 8), SPS is not active in the system studied. The SPS does not af- fect the plaque-forming capacity of a d+ variant (the Mahoney strain) either at high or at low concentration of bicarbonate. Thus, SPS is a major factor in t.he manifes- tation of the d marker of poliovirus. A question may be raised whether or not the different effect of attenuated and pathogenic strains of poliovirus upon the human organ- ism (or the monkey) is associated with the presence of some compounds related to SPS (e.g., heparin, hyaluronic acid). This ques- tion is important-the more so as, for ex- ample, heparin was found to have a SPS-like effect upon the multiplication of encephalomyocarditis virus (4)) and hy- aluronic acid inhibited the cytopathic action of poliovirus (9).

REFERENCES

I. VOGT, M., DULBECCO, R., and WENNER, H. A., Virology 4, 141-155 (1957).

2. TAKEMORI, N., and NOMURA, S, Virology 12, 171- 184 (1960).

Y. AGOL, V. I., and CHUMAKOVA, M. YA., Voprosy Vimsologii 6, 617-619 (1961).

&. TAKEMOTO, K. K., and LIEBHABER, H., Virology 14,456-462 (1961).

’ A sulfated polysaccharide that inhibited multi- plication of a mutant of encephalomyocarditis vi- rus, was recently isolated from agar also by Take- moto and Liebhaber (4) by a somewhat different procedure. The same authors (5) suggested that the action of SPS could be blocked by the addition of some polycations to the overlay.

DISCUSSION AND PRELIMINARY REPORTS 223

6. LIEBHABER, I-I., and TAKEMOTO, K. K., Virology 14,502~504 (1961).

6. HSIUNG, G. D., and MELNICK, J. L., J. Zm- munol. 80,282-293 (1958).

7. AGOL, V. I., and CHUMAKOVA, M. YA., Acta Viral. (Prague) 6, 2431 (1962).

8. AGOL, V. I., and CHUMAKOVA, M. YA., Voprosy Virusologii 5,492-493 (1960).

9. SALGANIK, R. I., SHCHERBATYKH, V. P., and PROTAS, L. K., Abstracts of Papers Presented

at the XIVth Meeting of Ivanovsky Institute

of Virology, Moscow, 1961, pp. 52-53.

V. I. ACOL M. YA. CHUMAKOVA

Institute of Poliomyelitis and Viral Encephalitides

U.S.S.R. Academy of Medical Sciences Moscow, U.SS.R.

Received January 26, 1962