DISCUSSION AND PRELIMINARY REPORTS 321

Cytoplosmic Crystals in FL Cells Infected with an Unclassified Enteric

Virus (Presumably a New Type of ECHO)

Since crystalline arrays of poliovirus and Coxsackie virus particles were demonstrated in sections of cultured cells by electron microscopy (l-1), attempts have been made to observe other enteric viruses by similar techniques.

Two freshly isolated strains of an enteric virus believed to be ECHO1 were found to produce cytoplasmic crystals in FL amnion cells, and this is thought to be the first demonstration of ECHO virus crystals in the cell cytoplasm.

The virus was isolated in tissue cultures from stools of two different patients (I’. R. and T. B.). The P.R. strain was isolated in FL and primary amnion cell cultures and human uterine tissue in plasma clot cultures. Monkey kidney cells were susceptible to infection by the virus after passage in amnion or uterine cultures. The T.B. strain was isolated from the stool suspension in monkey kidney and human uterine cultures. Although isolation in FL cells was not attempted, this cell line was readily susceptible to the virus passed from monkey kidney cultures. Infant mice and adult mice were insusceptible to infection with both strains. Neutralization was not obtained with antisera for all known cytopathic enteroviruses. Pooled human gamma globulin had some neutralizing activity and there was more than a fourfold rise in the neutralizing titer when the patient’s acute and convalescent sera were compared.

The P.R. and T.B. strains were inoculated into cultures of FL cells (5) with the technique described for poliovirus (1). Cultures were harvested at various times after infection and prepared for electron microscopy. In those harvested when cytopathic changes in the few cells still attached to the glass wall were seen by light microscopy, par- ticles in crystalline array were observed in the cytoplasm by electron microscopy (Figs. l-3). The crystals of virus particles were surrounded by vesicles of the endoplasmic reticulum. The size of the crystals varied, some were as large as 1.7 p X 1.0 I.C. The center-to-center measurement of particles in the best linear patterns was 21 rnp; individual particles measured up to 17 rnp. Considering the shrinkage in preparation, the actual particle diameter is presumably larger. Figure 3 shows a crystal of virus particles in close apposition to endoplasmic reticular membranes with aligned ribonucleoprotein (RNP) particles. Such spatial relation-

Supported by grants from The National Cancer Institute, Public Health Serv- ice, and The American Cancer Society.

1 This virus has not yet been submitted as a candidate new ECHO type to t.he Committ,ee on the Enteroviruses of the National Foundation.

322 DISCUSSION AND PRFLIMIN4RY REPORTS

FIG. 1. Virus particles (P.R. strain) as crystalline arrays and chains between cytoplasmic vesicles of an FL cell. Magnification: X 80,000.

DISCUSSION AND PRELIMINARY REPORTS 323

FIG. 2. Crystalline array of virus particles in the cytoplasm of an FL cell from culture inoculated 20 hours previously with the enteric virus (P.R. strain); x 75,ooo.

FIG. 3. An ordered array of enteric virus particles (T.B. strain) with adjacent ergastoplasmic membranes and particles. Plasma membrane, PM; chromatin, CHR; nucleolus, NCL; virus crystal, VC. X 75,000.

324 DISCUSSION AND PRELIMINARY REPORTS

ship and similarity between virus and cytoplasmic RKP particles have also been observed in poliovirus- and Coxsackie virus-infected cells (6). Other changes in the nuclear and cytoplasmic components of cells infected with these two strains of enteric virus were in most respects similar to those observed in cells infected w&h poliovirus and with Coxsackie viruses.

REFERENCES

1. ST~JART, D. C., JR., and FOGH, J., Exptl. Cell Research 18, 378-381 (1959). 2. FOGH, J., and STUART, II. C., JR., Virology 11, 308-311 (1960). 3. MORGAN, C., HOWE, C., and ROSE, H. M., Virology 9, 145-149 (1959). 4. FOGH, J., and STIJART, I). C., JR., Virology 9, 705-708 (1959). 5. FOGH, J., and LVND, R. O., Proc. Sot. Exptl. Riol. Med. 94, 532-537 (1957). 13. FOGH, J., and STUART, I). C., JR., Federation Proc. 19, 404 (1960).

Division of Laboratories and Research,

New York State Department of Health, Albany, New York

Received July 18, 1960

DONALD C. STUART, JR. J~RGEN FOGH~ HILDEGARD PLAGER

Studies of the Virus of Foot-and-mouth Diseose: A Relationship between

Complement-fixing Activity and Neutralization of Infectivity

Although both the complement,-fixation t’est and the neutralization t,est yield valuable information on virus-antibody interactions, little at,tention has been given to the relationship between the results ob- tained by the two tests when applied simultaneously to a single series of virus-antibody mixtures.

The experiments reported here were made during the course of investigations of the inhibition of virus-antibody complex formation by excess of either reactant. Reaction mixtures of complement, anti- serum and virus were prepared and incubated at 37” C for half an hour in a manner similar to that described elsewhere (1). A modified hemolysis depression procedure was employed in which each reaction mixture contained one 70 % hemolysis unit of complement. Before addition of the hemolytic indicat’or and complet.ion of the complement- fixation t,est, a portion of the reaction mixture was removed for imme- diate titration in unweaned mice (2) and on monolayers of pig kidney cells (3). Essentially similar result’s were obtained by both titration procedures. Thus, parallel observations of complement-fixing activity and infectivity were made on the same reaction mixtures. The clarified, inactivated, hyperimmune guinea pig sera were employed at dilutions

* Present address: Roswell Park Memorial Institute, Buffalo, New York.