poliomyelitis - journal of bacteriologysome properties of poliomyelitis virus' paulf. clark,...

SOME PROPERTIES OF POLIOMYELITIS VIRUS'PAUL F. CLARK, JOHN SCHINDLER AND DAVID J. ROBERTSDepartments of Patholgy and Bacteriology, University of Wisconsin, and

Bacteriology and Hygiene, University of ChicagoReceived for publication May 24, 1930

In view of the general lack of confidence either in the "globoidbodies" of Flexner and Noguchi or the streptococci of Rosenowas the specific etiologic agents in poliomyelitis, it would seem thatsome progress might be made by an indirect attack, such as thatoffered by the possibility of concentrating the virus by distillationin vacuo, or by some method of chemical precipitation. Eitherof the organisms mentioned, if of etiologic significance, must bepresent in the "filtrate" which is so uniformly infective. If, then,we can concentrate 50 or 100 cc. of this filtrate to 1 cc., the relativeincrease in the number of organisms shouldmake them more easilycultivable (Bloomfield and Felty, 1924) and more easily demon-strable by direct microscopic examination. Also, the confusingextraneous material present in a brain cord suspension should bereduced by this procedure, and such a "concentrate" might bemore effective as an artificial immunizing agent than the suspen-sions used heretofore.

In the summer of 1912, Flexner, Clark and Frazer employedthe method of distillation in vacuo at relatively low temperatures,200 to 300C., for the purpose of concentrating filtered washingsfrom the nasopharynges of "contacts" with cases of poliomyelitis.That this method did not seriously injure the virus which mightbe present in the throats, was shown by subjecting the regularsalt solution poliomyelitis filtrate to the same procedure. Mon-keys injected with brain-cord filtrate which had been concentrated

1 This work was made possible through aid received from the InternationalInfantile Paralysis Committee from funds given by Jeremiah Milbank.

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214 P. F. CILARK, J. SCHINDLER AND D. J. ROBERTS

about four or five times by volume, came down typically withcharacteristic symptoms and lesions. The results with the con-centrated nasal washings were negative, however, and sincea "carrier" of poliomyelitis virus (Flexner, Clark and Frazer,1913) was successfully demonstrated by injecting small amountsof the original washings into the sheath of the sciatic nerves andlarge volumes (140 cc.) intraperitoneally, this in vacuo method wasnot published at the time.

Subsequently, Amoss and Taylor (1917) used this procedure inconcentrating distilled water nasal washings from normal personsand from cases of poliomyelitis, demonstrating therewith thatthese washings, especially those from normal individuals, fre-quently had the power of inactivating poliomyelitis virus.The method of distillation in vacuo hardly needs description

save to mention one or two minor details which We have foundhelpful in applying the method to this material. Many sealedcapillaries in the spherical flask containing the virus to be distil-led prevented excessive bumping. Circulating ice water in thedistilling tubes aided in maintaining low temperature during thewarmer months; two smaller flasks connected in series with thedistilling flask, and each separated by an air tight valve made itpossible to add an indefinite amount of material without break-ing the vacuum. Thus the process could be run continuouslyuntil the desired concentration was effected. By using new rub-ber fittings every two or three times and paraffin on the joints,a vacuum of 12 to 15 mm. of mercury was obtained with the waterpump. For the rapid progress of the concentration, it is neces-sary to apply the heat rather continuously, so a double jacketedwater bath with an electric heater and thermostatic control wasemployed. The temperature outside the virus flask has been keptat about 400 while the inside temperature has remained usuallyat 200 to 21°C., never going above 25°C. The whole apparatusis assembled and sterilized in the autoclave before using. It isreadily possible to concentrate 500 to 600 cc. of the virus filtrateto one-fiftieth or one-sixtieth of the original volume in four tofive hours.

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PROPERTIES OF POLIOMYELITIS VIRUS

CONCENTRATION OF THE VIRUS BY DISTILLATION IN VACUO

As we intended to concentrate the virus to a greater degree thanin 1912, we at first employed in different experiments, distilledwater, twentieth physiological saline or tenth physiologic salineas the diluent for the brain and cord suspension. This was toavoid possible injury to the virus through the marked increasein sodium chloride content coincident with the decrease in volumeof water. We succeeded in obtaining active filtrates with thesediluents, but the small amount of electrolyte in the suspensionscaused such persistent turbidity, as to reduce the amount of virusthat would pass through a Berkefeld or Mandler Filter as com-pared with that which would pass when physiologic saline wasemployed (see table 1, series 1). It was possible, however, todemonstrate that a filtrate made from a 10 per cent suspension ofglycerinated poliomyelitis brain and cord diluted with twentiethphysiologic saline could be reduced in volume by distillation invacuo to one thirtieth of the original volume and produce thedisease when injected in the amount of 0.03 cc. The control ani-mal died of colitis so that no quantitative statement can be madewith reference to this series (see table 1, series 2).

Because, however, of the relative lack of potency of the filtratesmade with low salt concentrations, all of the later experimentswere carried out with physiologic saline (0.85 per cent). Series3 (table 1) shows the results of such a reduction in volume inwhich the "concentrate" was at least five or six times as potentas the filtrate and apparently as potent as the original brain cordsuspension. The "concentrate" obtained by reducing the watercontent to one-thirtieth or less of the original volume is a light,reddish brown, thick, viscous fluid from which a small amount ofprecipitate gradually settles out leaving a moderately clear lightamber colored fluid.

In the fourth series, the concentration procedures gave a residueeven more potent than the original 5 per cent brain cord suspen-sion (see table 1, series 4).The results have not in every instance been successful, however,

especially in the last six months when the virus seems to have

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216 P. F. CLARK, J. SCHINDLER AND D. J. ROBERTS

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PROPERTIES OF POLIOMYELITIS VIRUS 217D -o0+ - I So3 I

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altered in potency. Whether this has been due to the varioustypes of handling to which the virus has been subjected or to oneof the spontaneous changes in virulence described by Flexner,Clark and Amoss (1914) is not clear. During this period, doses aslarge as 2 cc. of the filtrate have sometimes failed to produce thedisease and when effective doses have been given, the progress ofthe disease has been slow, and recovery would have been success-ful'in many of the animals had they not been killed with chloro-form. Also, the individual susceptibility of an animal relativelymore resistant than human beings, becomes more apparent whenone is dealing with doses approximating the minimal effectiveamounts. One is reminded of the markedly irregular resultsobtained by the injection of the globoid bodies (Flexner, Noguchiand Amoss, 1915) and of the occasional successful infectionfollowing the injection of very small amounts of the filtrate.

VIABILITY OF CONCENTRATED VIRUS IN SATURATED SALT

SOLUTIONS

Since the saturated salt solution resulting from the evaporationof the water did not appear to injure the virus immediately, itseemed advisable to determine the viability of the virus underthese conditions so generally destructive for living cells. Withthe "concentrate" maintained at refrigerator temperature (10° to120), the virus remained fully potent for at least two months, be-coming somewhat less active but still highly infective at the endof four or five months (see table 2, series 5 and 6).

Streptococci isolated by Dr. E. C. Rosenow from cases of polio-myelitis were treated in the same way as the virus, save that normalrabbit brain and cord were used to make the suspension and to the300 cc. of filtrate were added three blood agar slants of theorganisms suspended in 1.5 cc. salt solution. The whole wasconcentrated 30 to 1 and cultures made at intervals. Growthwas obtained, following the transplantation of liberal quantitiesthrough the fifteenth day but not on the thirty-fifth day.

It is possible that the remarkable resistance of the poliomyelitisvirus to the lytic effect of the saturated saline solution may be due

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to the protective action of the associated proteins as shown byOlitsky and Boez (1927) for the virus of the mosaic disease oftobacco. Yet in the same medium, streptococci are destroyedmuch more promptly. They become shrunken and granularwithin three weeks.When considered J comparison with the prolonged viability

of the virus under similar conditions, this would tend to indicatethe absence of causal relation between the streptococci and polio-myelitis.

ATTEMPTED CULTIVATION AND MICROSCOPIC EXAMINATION

Many attempts have been made to ci4tivate organisms fromthe concentrates, and later from the dialyzed concentrates, and tostudy smears stained by a variety of methods. The mediaemployed were glucose brain broth, Smith-Noguchi medium,blood agar, Locke's egg medium, ascitic fluid broth and agar andordinary beef infusion agar and broth. All told, about twentyconcentrates from thirty monkeys have been planted and subse-quently examined for growth. Occasionally contaminationshave been found, staphylococci, a diphtheroid, aerobic sporeformers and finally, in one instance only, a streptococcus. Thisstreptococcus was of the viridans type and fermented lactose,mannitol and salicin. It produced no apparent symptoms in amonkey when one-half of a blood agar culture was injecteddirectly into the brain substance.

Direct microscopic examination of the concentrates, using avariety of stainiing technics, has shown nothing that has not beenfound in normal brain cord suspensions treated in the same way.If streptococci were etiologically related to poliomyelitis, weshould expect to find them in smears and cultures from thesehighly concentrated filtrates.

PRECIPITATION OF THE VIRUS WITH VARIOUS PROTEIN

FRACTIONS

a. Water soluble and water insoluble fractions

The presence of the large amount of salt in the concentrateproved, however, to cause difficulty in the examination of the

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PROPERTIES OF POLIOMYELITIS VIRUS 225

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smears and was obviously also a handicap to its use in animalsunless redilution were practiced.

Hence, the concentrate was dialyzed with aseptic precautionsagainst sterile distilled water in autoclaved bags made with celloi-din dissolved in equal part of alcohol and ether. Later in thework, cellulose sausage casings made by the Visking Corportion ofChicago were employed. The dialysis was continued until thediffusate no longer gave a test for chlorides upon the addition ofsilver nitrate solution. With the loss of salt, a rather heavybrownish-green precipitate was thrown out of solution and easilyremoved by centrifugation leaving a clear, straw-colored super-natant.The precipitates, secured in this manner from three different

concentrates made from filtrates from six animals, have beentested for virus. After three washings in distilled water, the sedi-ment has been taken up in a small volume of physiologic salinein which it is largely redissolved and amounts equivalent to asmuch as 140 cc. of the original filtrate have then been injectedintracerebrally into monkeys. In every instance the animalshave remained entirely well (see table 3, series 7, 8 and 9). Theclear supernatant on the other hand has in every case provedeffective in producing the disease, although in one instance(monkey 46) the animal did not become definitely paralyzed.A preliminary but not precise titration of the dialysate indicates

that there is little loss of potency during the process, due allow-ance being made for the increase in volume (table 3, series 10).The diffusate, on the other hand, after the complete dialysis

of the concentrate, has in one instance been concentrated andsubsequently dialyzed free from sodium chloride. This materialwas injected into monkeys without producing poliomyelitis(table 3, series 11).

It would seem reasonable to conclude therefore that the virusis not diffusible through a celloidin membrane, is not present ineffective amounts in the water-insoluble proteins even when theprecipitate from a volume of concentrate equivalent to 140 cc. ofthe original filtrate has been injected, and that it does remainconstantly associated with the water soluble proteins.

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Further evidence along this line was sought by precipitatingboth the dialyzed concentrate and the regular 5 per cent filtratewith ammonium sulphate.

b. Fraction precipitated by half saturation with ammoniumsulphate

After the water insoluble fraction had been removed from thedialysate by centrifugation, a grayish precipitate was secured byadding an equal volume of sterile saturated ammonium sulphatesolution. This precipitate was removed by centrifugation andwashed three times with half saturated ammoniium sulphate solu-tion. This washed sediment was then taken up in a small volumeof distilled water and dialyzed against sterile distilled water untilthe diffusate showed no trace of sulphates upon the addition ofbarium chloride solution and the dialysate no odor of ammoniaupon the addition of strong sodium hydroxide to a test portion.A similar procedure was carried out with the virus filtrate.In both instances, the animals injected with the dialyzed precipi-

tate obtained from the dialysate came down typically while oneof the animals (60) injected with the similar material obtainedfrom the filtrate came down typically and the other (74) developeda definite tremor, excitability and ataxia on the ninth day but thedisease did not progress. Seven weeks later this animal devel-oped poliomyelitis following the injection of concentrate. Ineach of these instances the equivalent of relatively large amountsof virus was injected and no attempt has as yet been made toobtain quantitative information (table 3, series 12).

c. The fraction precipitated by complete saturation with ammoniumsulphate

To each 100 cc. of the supernatant secured after half saturationas described above, were added 35 grams of sterile ammoniumsulphate. After shaking the mixture and-allowing it to stand forfifteen to twenty minutes, a light flocculent precipitate appearedwhich could be removed only by prolonged centrifugation (thirtyto sixty minutes). This precipitate was then washed three times

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with saturated ammonium sulphate and was resuspended insterile distilled water and dialyzed until the diffusate showed notrace of sulphates.The results after inoculating this material were at first con-

fusing. In two series, the animals (51 and 67) that received thisalbumen fraction from the dialysate, did not develop poliomyelitiswhile in the same two series, the animals that received the similarfractions from the filtrate developed typical poliomyelitis. Themost plausible explanation of the difficulty seemed to be themarked difference in the volumes employed and the resultingdifference in the effect of centrifugation. Two hundred cubiccentimeters of filtrate had been employed whereas only 25 to 50cc. of the dialysates had been employed. This fact made it pos-sible to remove the precipitate resulting from half saturationwith ammonium sulphate more completely in the case of the dialy-sate than in the case of the filtrate. Accordingly, in the next se-ries, prolonged centrifugation was used following the half satura-tion of the filtrate before completely saturating the supernatantwith ammonium sulphate. The precipitate so secured did notproduce poliomyelitis whereas the half saturation precipitatedid (monkey 75). At the same time the half saturation portionof the dialysate was less completely centrifuged than usual so thatthe supernatant was not completely clear. Following this pro-cedure, the fraction obtained by complete saturation did producepoliomyelitis in a monkey (see table 3, series 13 and 14).The final supernatant after the last precipitation by complete

saturation, was also tested in two series for the presence of thevirus and in neither instance did it produce any effect in animals.This supernatant was dialyzed free from ammonium sulphateand then concentrated to a few cc. and injected (see table 3,series 15).There seems little doubt therefore that the virus is precipitated

with the globulin fraction by ammonium sulphate and that asthe earlier dialysis experiments indicate, the pseudo or watersoluble globulins are the essential carriers of the virus.

In one instance each, the precipitates obtained by the additionto the virus of two volumes of absolute alcohol in the one case and

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three volumes of acetone in the other, produced no result wheninjected intracerebrally into monkeys.

USE OF CONCENTRATION IN VACUO IN DETERMINING THE INFEC-TIVITY OF FECES AFTER FEEDING MONKEYS WiTH THE VIRUS

The demonstration by Aycock and others that poliomyelitismay apparently be spread through milk supply, raises the ques-tion whether the milk is contaminated by droplets from the naso-pharynx or whether the virus may occasionally pass through thefeces unaltered and thus reach the milk supply. Also, the possi-bility that the infection may in these milk borne epidemics actu-ally take place through the stomach or the intestines rather thanthe nasopharynx is again suggested.

Accordingly, 3 monkeys were fed about once in five days bystomach tube with 20 to 25 cc. brain cord suspensions, two for aperiod of fourteen weeks with a total of 310 cc. each and one foreight weeks with a total of. 215 cc. These animals did not developpoliomyelitis and subsequently all came down typically follow-ing intracerebral injection of the virus. This is in accord withearlier observations by Flexner and Lewis (1910), and with a morerecent report by Schultz, (1929). Leiner and v. Wiesner, throughthe use of sodium bicarbonate to neutralize the hydrochloric acidof the stotnach and large doses of morphia to reduce the peristal-sis, succeeded in producing the disease in 2 out of 5 monkeys fed,but Amoss (1928) following their procedure and giving evenlarger doses did not confirm their observations.During the course of this feeding experiment, after the animals

had been fed a total of 150 cc. each over a period of approximatelya month, they were, after two successive feedings of 20 cc. each,placed at once in clean, sterile cages. The feces of the three mon-keys were collected each time over a period of forty-eight hours,resulting in a total of 65 grams. A 10 per cent suspension wasmade, shaken, centrifuged and the supernatant filtered throughsterile Mandler filters. The 500 cc. of filtrate obtained wereconcentrated in vacuo to 10 cc., and dialyzed free of sodium chlo-ride, thus being increased in volume to 30 cc. One and one-fourthcubic centimeter of this dialysate, representing roughly the salt

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PROPERTIES OF POLIOMYELITIS VIRUS 231

solution soluble equivalent of the feces passed by one monkey inabout twelve hours, was injected intracerebrally into monkey 39.Following an incubation period of six days, the monkey developedtypical symptoms of poliomyelitis which became progressivelyworse, with complete paralysis and a fatal termination two dayslater. The identity of the disease was confirmed by the micro-scopic findings and by reinoculation into another monkey withthe production of typical poliomyelitis in that animal also (seetable 4, series 16).

TABLE 4

Infectivity of feces after feeding monkeys with the virusSee table 1

m ~~~~~~~~~~~~~~~~~~~PRE-R SOURCE OF INUU AMOUNT DATE OF SUBSEQUENT VIOUSM MATERIAL INJECTED INOCULATION COURSE HIS-0 TORTY

39 A variety Dialyzed con- 1.25 cc. rep- 23-III-29 Paralyzed Noneof suspen- centrate of resent- 30-III-29sions fed feces from ing feces Deadto monkeys monkeys fed passed in 31-III-2919, 21, 30 with virus about 12

hours16,

54 39 Suspension of 1.5 cc. 31-V-29 Paralyzed Nonebrain cord 7-.VI-29

Prostrate8-VI-29Killed11-VI-29

In five series of experiments with a total of ten monkeys in-jected, we have tried to repeat this experiment without, however,using the month's preliminary feeding which seemed to us notinvolved in the immediate question. The duration of time offeeding of the virus prior to the beginning of collection has variedfrom thirty minutes to four days; the duration of time of collec-tion after feeding has varied from sixteen to forty-eight hours;the virus has also been fed during the period of collection inamounts from 20 to 150 cc. in twenty-four hours; suspensionsfrom glycerinated and from fresh tissues have been employed; and

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the dialysates have been injected not only intracerebrally involumes of 1 to 3 cc. but intraperitoneally in volumes of 2 to 10 cc.All of these have given negative results; so we are forced to con-clude that the virus does not pass through the intestinal tract ofmonkeys even when fed in enormous amounts save under excep-tional and unknown conditions. Also, feces collected from 3groups of monkeys (8 in all) during the acute stages of the diseasewere collected and carried through the same procedure. Thethree monkeys inoculated with these dialyzed concentrates, re-mamned well. By exclusion, the importance of the upper respira-tory tract in the transmission of poliomyelitis is again emphasized.

SUMMARY AND CONCLUSlONS

It has been possible to concentrate poliomyelitis virus by dis-tillation in maCuO so that the residue is more potent than the origi-nal filtrate or the brain cord suspension from which the filtratesare made.The virus remains potent in the "concentrate," even though

the solution contains sodium chloride to saturation, for more thanfour months.On the other hand, streptococci, isolated from cases of polio-

myelitis, concentrated in a filtrate of normal brain cord suspen-sion, are no longer viable after thirty-five days. This would tendto discredit the streptococcus as the specific etiologic agent inpoliomyelitis.The "concentrates" can be dialyzed against sterile distilled

water without essential loss of potency.The virus is present in the water-soluble portion of these dialy-

sates and not in the water-insoluble or euglobulin fraction.The virus is not demonstrable in the concentrated diffusate.The virus is present in the globulin precipitate, obtained

either from the regular filtrates or from the "dialysates," followinghalf saturation with ammonium sulphate.Complete saturation of the thoroughly centrifuged supernatant

so obtained, gives a precipitate (albumen fraction) which does notcontain the virus.

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The virus is not demonstrable in the concentrated final super-natant after complete saturation with ammoniumn sulphate.

These fractional precipitations together with the facts obtainedfollowing dialysis indicate that the virus remains constantly asso-ciated with the pseudoglobulin fraction of the proteins.

In one out of six experiments, the feces, obtained following thefeeding of large volumes of poliomyelitis virus, gave an inoculumwith which the disease was experimentally produced. Theconcentration of feces from acute cases gave negative results inthe three groups investigated. These experiments would indi-cate that the virus does not commonly pass through the intestinaltract of monkeys in an infective form.Many attempts have been made to cultivate a significant organ-

ism from the highly infective concentrates and dialysates withnegative results. Streptococci were found as a contaminant inone instance only. Stained smears have also shown nothing thatcould not be found in the "dialysate" from normal sources.

REFERENCES

AMOSS AND TAYLOR 1917 Jour. Exper. Med., 25, 507.AMOSS, H. L. Filterable Viruses, Rivers et al., Williams and Wilkins, Balti-

more, 1928, 174.BLOOMFIELD AND FELTY 1924 Jour. Exper. Med., 39, 367.FLEXNER, CLARK, AND Amoss 1914 Jour. Exper. Med., 19, 195.FLEXNER, CLARK, AND DOCHEZ 1912 Jour. Amer. Med. Assoc., 59, 273.FLEXNER, CLARK, AND FRASER 1913 Jour. Amer. Med. Assoc., 60, 201.FLEXNER AND LEwis 1910 Jour. Exper. Med., 12, 227.FLEXNER, NOGUCHI, AND Amoss 1915 Jour. Exper. Med., 21, 91.LEINER AND V. WIESNER 1910 Wien. Klin. Woch., 23, 91.OLITSKY AND BOEZ 1927 Jour. Exper. Med., 45, 815.SCHULTZ, E. W. 1929 Proc. Soc. Exper. Biol. and Med., 26, 632.

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