From the Laboratories of the Rockefeller Institute for ~r Research. New York.

The Adaptation of Poliomyelitis Virus, Lansing Strain, to Newborn Mice and the Use of Their Central Nervous Tissues for Preparation of Complement-fixing Antigen*.

By Jordi Casals and Peter K. Olitsky.

The pathogenic action of neurotropie viruses for the albino mouse depends on several factors, one of which is the age of the animal. I t has been found that with a number of such viruses young mice are more susceptible than older ones, especially after the infective agents are in- troduced into nonneural (peripheral) tissues. The earlier work in this laboratory showed that this was true if the age of the animals employed in the experimental work was 7 to 9 days for rabies and 12 to 14 days for other neurotropie viruses (Olitslsy, Sabin and Cox, 1936; Sabin, I940; Morgan, I941; Olitslcy and Harford, 1938, and Casals, 1940). An elabora- tion of this finding by several investigators studying additional neurotropic viruses revealed that even younger animals, 3 to 8 days of age, exhibit the greatest susceptibility to extraneurally inoculated viruses such as those of Bwamba fever, herpes simplex, St. Louis eneephMitis, West Nile disease, Japanese B encephalitis and Western and Eastern equine ence- phalitis (Lennette and Koprowski, 1944). Of interest is the recent dis- closure that the Coxsaekie group of viruses are pathogenic for newborn mice, with some of the strains active by both neural and nonneural routes of administration and some of them not at all reactive in weaned animals (Dalldor[, 1950; Melnick, 1950).

One noteworthy exception to this rule of increased susceptibility of young mice to neurotropie viruses is the Lansing-type, or rodent-adapted strain, of poliomyelitis virus. In this instance the youngest animals are, conversely, resistant to the virus, and the resistance decreases as the age

* Dedicated to Professor Robert Doerr on his 80th birthday with our reverence and esteem.

The adapta t ion of Poliomyelit is Virus to new born mice. 453

of the mouse increases f rom 1 to abou t 21 days ; a t t h e l a t t e r t ime is no t ed the full response as is seen in a d u l t mice (Sabin, 1950; Dalldor], 1950; Casals and Olitsl~y, unpub l i shed work).

F o r reasons to be given in the nex t section, the a d a p t a t i o n of a Lansing- t y p e vi rus to young mice was desired. F o r th is purpose the ~ E F 1 s t ra in of po l iomyel i t i s v i rus was used. I t is one of the few known r o d e n t - a d a p t e d s t ra ins and was d iscovered in th is l a b o r a t o r y in 1943 (Schlesinger, Morgan a n d Olitsl~y). This v i rus conforms to t he original Lans ing type , the Arm- s t rong s t ra in (Armstrong, 1940-1941), in a l l respects except t h a t in mice i t p roduces a more un i form reac t ion and a higher in fec t iv i ty t i t e r (Yager, Olitsl~y and Lahelle, 1949). I n j e c t e d in t r ace reb ra l ly in to newborn mice of the W-Swiss s t ra in , 1 to 4 days old, cer ta in in jec ted animals would reac t wi th neurological signs following the in t roduc t ion on ly of h ighest concentra- t ion, 10 -1 , of the virus. The signs compr ised general ly , f laccid para l - ys is of a n y one or al l of the ext remit ies , t e rmina t i ng as a rule in d e a t h .

As the fol lowing will show wha t appea r s to be a va r i an t of the vi rus has been induced b y the a d a p t a t i o n of the MEF1 s t ra in to newborn mice. The resul t has been to secure a s t ra in which is regu la r ly and un i fo rmly pa thogen ic to newborn mice which are to a re la t ive degree n a t u r a l l y res i s tan t to t he s t anda rd , u n a d a p t e d s t ra in . The disease now p roduced in the newborn wi th th is a d a p t e d vi rus shows a cons iderab ly shor t e r i ncuba t ion pe r iod a n d an in fec t iv i ty t i t e r of the t issues of the cen t ra l ne rvous sys t em of af fec ted newborn t h a t is h igher t han the t i t e r ob ta ined wi th s imi lar adu l t mouse tissue.

Materials and Methods. Virus.--In the earlier experiments a t tempts were made to adap t the

Armstrong-Lansing strain to newborn mice; when, however, i t appeared tha t the MEF1 virus possessed a greater pathogenic power, the la t ter was sub- s t i tuted. The s tandard strain was ordinari ly propagated in 3- or 4-week old mice and the number of passages in mice tha t this MEF1 virus had undergone since its isolation in 1943 was probably not more than 40; the LDs0 t i ter in adul t mice of the s tandard Armstrong-Lansing virus was 3.5 or less, and tha t of the ]KEF1 strain, 4 or less, reciprocal log units.

The Armstrong strain of virus, obtained from 3- to 4-week old mice which were paralyzed following the inoculation of this virus and made into a 10 -1 suspension in physiological saline solution or 0.2 per cent bovine plasma albumin (Olitslcy, Yager and Murphy, 1950) was t i t r a ted by intra- cerebral injection into mice of different ages. In animals 21 days old or older the infect ivi ty t i ter ranged from 10 -3.5 to 10 -3"~ or a geometric mean of 10 -3. In the newborn the t i ter was not only much lower, about 10 -1"5, but the incubation period was considerably longer and several mice remained well, free from any signs. A similar picture was seen when the MEF1 strain, instead of the Armstrong, was injected. Thus these findings confirmed the prior results and showed the relative insusceptibil i ty of newborn mice to Lansing-type viruses.

454 J. Casals and P. K. Olitsky:

Mice.--Thc W-Swiss strain of mice (Webster, 1939) was used throughout these experiments. They were secured from a single dealer and were mated in this laboratory at the ages of 2 or 3 months. The newborn were employed, as a rule, when they were 1 t o 4 days o l d .

Other methods applied i n this study, the preparation of virus suspensions and their inoculation, and the general procedures, have already been described in detail (Casals, Olitslcy and Anslow, 1951 a):

Adaptation o/ MEF1 Strain to Newborn Mice.--Serial passages in new-

born mice, 3 or 4 days of age, as a rule, were carried out by means of in t ra-

cerebral inoculat ion. Brain and c o r d suspensions prepared f rom the in-

jec ted newborn mouse tha t became para lyzed in the earliest passages

comprised the source mater ia l for the serial transfers. The series was

cont inued unt i l the s ixth to the n in th passage when " b l i nd" passages

were carried out, i. e., central nervous tissue from inocula ted mice was

r emoved for t ransfer on the th i rd day, even though this was still in t he

incubat ion period when animals were apparen t ly normal . B y the t e n t h

passage, about half the n u m b e r of inocula ted newborn were para lyzed

or succumbed to the specific infect ion; from the e leventh to the twen t i e th ,

90 per cent ; f rom the t w e n t y first to the th i r t ie th , 98 per cent and a t

T a b l e 1.

No. of passages in newborn mice

Inoculation of 10 -1 dil. I~IEF1 virus into 3--4 day old mice

~ of reactors Average in-

cubation period (days)

LDso titer in adult mice (reciprocal of log)

0 (standard) 73 11.7 3.5 • 0,447*

85 100 100 97

100 99 96

100 99

98 100 100 100 100

9 13 15 23 28 32 36 38 53

8.6 5.3 6.2 5.7 3.7 4.1 6.7 4.7 3.7

3.2 2.3 2.4 2.3 2.5

58 72 75 76 77 77 (a)

* Mean geometric value of 30 titrations.

3.9 4.6 4.5 4.8 3.8 4.0 4.0 4.0

Mean 4.2 ~: 0.374

5.0 5.1 6.1 5.6 5.2 5.9

Mean 5.5 • 0.454

The adaptation of Poliomyelitis Virus to new born mice. 455

present the seventy-seventh passage, 100 per cent (Table 1). I t would therefore appear that at some passage between the eleventh and twentieth a change was induced in the virus so tha t it became pathogenic for the natural ly resistant newborn mice.

This newborn-mouse adapted virus has now properties differing from those of the standard, adult-mouse strain. I t appears tha t at least a quanti- tat ive change has occurred, for the newborn-mouse adapted virus in a step-wise progression increased its infectivity titer, as tested in adult mice, from about 10 -~.5, the titer of the virus before adaptation, to more than 10 -5'0 , from about the fifty-eighth to the seventy-seventh, or present, passage in newborn mice, as shown in Table 1.

While the results given in the table relate to the outcome of infecti- v i ty titers as performed in adult mice, tests have revealed that the adap- ted MEF1 virus when t i t rated in newborn mice also showed an increased pathogenic effect. Thus, the unadapted strain exhibited an LDs0 titer of 2 or more log units higher in adult mice than in those 1 or 3 days of age; the adapted virus, however, in newborn mice was only about 0.5 log units lower than in mice 35 days of age.

Serial passage in newborn mice has brought about a variation in the standard MEF1 strain of poliomyelitis. The change was observed to pro- gress in a step-wise manner and was reflected by an increased pathogeni- city in the natural ly resistant newborn mice, from 1 to about 4 days in age. This, in turn, was revealed by a shortening of the incubation period of the experimental disease induced either in newborn or a.dult mice, espeeiMly in the former, from 11.7 to 2.3 days, and by more uniform and regular reactivity of the animals. Moreover, the adapted strain has in several repeated tests during various passages shown no immunological or serological deviation from the original, unadapted virus. Moreover, an additional property was acquired by the adapted strain, namely, the in- fectivity titer of the central nervous tissues of affected newborn mice be- came higher than those obtained from adult mice receiving the unadapted virus (Table 1). Finally, still another characteristic featurewas exhibited: central nervous tissues deriving from mice infected with it could now be successfully used in the preparation of an antigen for a complement- fixation test for poliomyelitis, a subject to be discussed in the next section.

Thus far a t tempts to adapt other strains of human poliomyelitis virus, the Brunhilde and the Leon (Bodian, Morgan and Howe, 1949) which are transmissible only to monkeys, have failed.

The Funct ion of I~lewborn-Mouse Adapted Virus as an Antigen for Complement Fixation in Poliomyelitis.

For the past 10 years this laboratory has been engaged in a t tempts to develop a specific complement-fixation test for poliomyelitis; the a t tempts

456 J. Casals and P. K. 01itsky:

failed. I t was bel ieved t h a t if the Lans ing - type virus were a d a p t e d to newborn mice progress migh t be made for the following reasons.

I n ear l ier tes ts i t was shown t h a t the J a p a n e s e B, and We s t e rn equine encephal i t i s viruses, b y serial passages t h rough newborn mice, developed no t only an increased pa thogen ic i ty for mice bu t also an increased infect- i v i t y t i t e r of thei r centra l nervous t issues dur ing the induced expe r imen ta l disease (Casals, Analow and Selzer, 1951). Since i t had been assumed tha t one of the factors needed for an effective pol iomyel i t i s an t igen is a sufficient concent ra t ion of i t per uni t volume of suspension, th is increased in fec t iv i t y t i te r could, therefore, be r ega rded as a po in t nearer the u l t i m a t e object ive. Indeed , i t was found t h a t the centra l nervous t issues of new- born mice could be emulsif ied in a smal ler volume of d i luent (1 : 1) t han could be those ob ta ined from adu l t mice (1 : 3).

The basis of this increased concentration of tissue or virus per uni t volume is supported by the observation of Folch (personal communication). The la t ter found tha t brain tissue deriving from 4-day-old mice had 87.8 per cent water and to ta l lipids, 3.5 per cent. Adult mouse brain showed 78.1 per cent water and 11.3 per cent lipids. Not only is the water content of newborn- mice brain higher but its l ipid content is much lower. The la t ter perhaps adding another favorable element since the preparat ion of the antigen is based on a method which reduces its l ipid content.

W h a t e v e r m a y be the exp lana t ion of the mechanism under ly ing the ant igenic ac t ion of newborn-mouse brMn and cord, these mate r i a l s can be used for the p repa ra t ion of a specific complement - f ix ing ant igen for pol iomyel i t i s v i rus (Casals and Olitsky, 1950).

Methods. The details of the preparat ion of the antigen with newborn- mouse adapted ~r virus, the method of preparing the reagents and eon- �9 dueting the test have a l ready been described (Casals, Olitsky and Anslow, 1951 b).

The points tha t should be emphasized are as follows. The preparat ion of antigen follows the method by which acetone-ether extracted central nervous tissues are used (Casals, 1949). Secondly, more potent antigens can be prepared from materials collected on the second day after inoculation of newborn mice, rather than on later days. Effective antigens have been made from the twenty first, or later, passage materiM. Thirdly, the average yield of antigen is 0,6 ml. per gm. of tissue. Large numbers of mice are there- fore necessary for preparation of even small amounts of antigen. Sera should be inactivated at 60 ~ C. for 20 minutes and 2 units of complement, preferably 1.8 units, are most satisfactory. Finally, and important, is the fact that control antigens should be used in each test. These are prepared in a way precisely similar to that employed for making the poliomyelitis antigen. The control antigens which serve to pick out anticomplementary and non- specific reactions, as commonly used in this laboratory, are those prepared with normal mouse brain and cord, or with Japanese B encephalitis and with Western equine encephalitis viruses, propagated in newborn-mouse brain and cord. A set up for the test includes invariably a known positive serum to test the activity of the antigens. Table 2 offers an illustration of an actual

The adaptation of Poliomyelitis Virus to new born mice. 457

test performed to determine the specificity and potency of an antigen pre- pared with newborn-mouse adapted ?r virus.

Table 2.

Antiserum

Mouse MEF1 . . . . . . . . . ,, Japan. B . . . . . . ,, West. equine. .

Cotton rat Armstrong. Monkey MEF1 . . . . . . .

,, Armstrong . . .

MEF1

Antigens

5apan. B. enc.

64* 0 0 128+ 0 0

64 0 8 0

16 0

West. equine enc. !

0 0

128+ 0 0 0

None

* 64 = reciprocal of highest dilution of serum giving a 2 + or better fixation.

Resul ts . - -With the newborn-mouse adapted MEF1-Lansing virus employed as antigen in complement-fixation tests, positive reactions were obtained from different animal species including mice and cotton rats immunized with MEF1 or Armstrong viruses, immunized and convalescent rhesus monkeys and chimpanzees and also man.

The fact that antigenic potency depends on the presence of newborn mouse brain is exemplified by absence of a potent antigen prepared from adult mouse brain in which newborn-mouse adapted virus was propagated.

The tests with monkey serum indicated that there is a possibility of cross reactions occurring between the MEF1 antigen and Brunhilde-virus produced antibody. This crossing was further noted in tests with human sera that were free from Lansing-type antibody, as determined by the virus neutralization test, and from whom the Brunhilde or a Brunhilde like virus had been isolated during the acute phase of poliomyelitis. This subject is still under investigation as is the range of practical application of the complement-fixation test to the problems of poliomyelitis. Further study is also considered of the occurrence of positive reactions at a certain level of dilution of serum in individuals who have recently been attacked by poliomyelitis and the relation of this level of reactivity with that existing in normal persons giving no history of a clinically apparent in- fection. Finally, the development of the complement-fixing antibody in poliomyelitis as it develops with the passage of time is also to be investig- ated.

D i s c u s s i o n .

Certain significant points stand out among the findings here reported which show the manner in which the standard MEFI virus developed its affinity for the nervous tissues of newborn mice. I t will be observed that the shortening of the incubation period and the enhancement of the in-

458 J. Casals and P. K. Olitsky:

fectivity titer as well as the uniformity of the response by the animals, all shown in Table 1, indicate thai the virus became adapted and the process was gradual. The fact that after adaptation the central nervous tissues could be used successfully as an antigen for a complement-fixation test for poliomyelitis added evidence for this adaptation. The finding that when fully adapted virus was passaged through adult mouse brain and the latter then failed to produce an effective complement-fixing antigen is consid- ered as a demonstration that the newborn nervous tissues provided the proper milieu for preparation of a potent an t igen- -a point supported by the different chemical composition of such tissue as compared with tha t deriving from adults.

I t is possible, therefore, that a variant strain has been brought for- ward by the continuous passages in newborn-mouse nervous tissues. This variant can be conceived as an adaptive variant rather than as a discontinuous one and is in accord with the recent concept as expressed by Ginsberg and Hors/all (1949), that a virus population can be considered as inhomogeneous, containing variant and parent viral particles. Here the majority of the particles are assumed to be adapted to adult nervous tissues (parent particles). Since newborn mice arc only relatively resistant the assumption is that a smaller number of the particles can attack the nervous tissues of both adult and newborn. Continuous serial passage in the newborn shifts numerically the viral particles towards those patho- genic for newborn mice (variant particles) with the result that the virus becomes pathogenic for them without loss of pathogenicity for the adult.

Summary. The manner has been described in which the iViEFI-Lansing type polio-

myelitis virus was adapted to newborn mice which to a relative degree are naturally resistant to the virus. This adaptation is manifested by a short- ening of the incubation period; uniform, regular and increased patho- genicity for the mice, and an enhanced infectivity of the central nervous tissues of the reactors. With this adapted virus which has the aspects of an induced, adaptive variant, it is possible to prepare an antigen for complement-fixation tests to detect poliomyelitis-virus infection. This test is specific and is applicable to the serum of several different species, including man, and may possibly reveal the existence of cross-reaction between two types of poliomyelitis virus, the Lansing and the Brunhilde. The range of its practical application to poliomyelitis problems remains, however, still for further study.

Acknowledgements. The authors are indebted for assistance to Major R. O. Anslow, U. S.

Army and Dr. G. Selzer, Cape Town Medical School, South Africa. For

T h e a d a p t a t i o n of Po l iomye l i t i s Vi rus to new b o r n mice. 459

s ~ m p l e s of s e r a t h e y .are g r a t e f u l to Dr s . A. B. Sabin, J. E. Melnick, D. Bodian, L. P. Gebhardt, J. E. Sallr T. Francis, J r . , G. C. Brown, G. E. Murphy , C. Rowe a n d t h e s t a f f of Willard Parker Hospital, N e w

Y o r k .

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