poliovirus officers POPULATION GENETICS APPLIED TO LIVE POLIOVIRUS

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					Here is an important paper on live poliovirus vaccine and its use in the community. This contribution will be of value for health officers and others who will be faced with decisions on immunization with live vaccine.

POPULATION GENETICS APPLIED TO
LIVE POLIOVIRUS VACCINE
Joseph L. Melnick, Ph.D., F.A.P.H.A.

NO VIROLOGIST ever works with a single viable virus particle-the best that he can do is to recognize the progeny of such an individual particle. Consequently it is only by studying populations that we can follow viruses as they infect, propagate, and become released from their hosts-whether these be simple tissue culture systems or human beings. In this discussion we shall not go into the developments leading to the present oral polio vaccines, as these have been well documented in recent years.1'2 Suffice it to say that all living things mutate, and that in passing virulent polioviruses through rodent CNS or primate tissue culture, at least five groups of investigatorss-7 showed that it was possible to select less virulent virus among the progeny. This work was brought to practical usefulness particularly by Sabin, who meticulously studied a number of progeny of single virus particles for neurotropism in monkeys, and finally selected three for small experimental trials in man.5 A number of investigators around the globe then participated in feeding these strains to large numbers-actually millions-of persons, and in studying the results.1'2 Such studies have included not only the human populations fed, but also the virus populations recovered from the vaccinees. As the vaccine virus is a
472

living organism which must multiply in order to immunize, it is essential to know as much as possible about the progeny viruses let loose in nature, and which still retain their property of infecting human beings. While this work was going on with the Sabin strains, a parallel series of studies was being carried out with the strains developed at Lederle Laboratories by Koprowski, Cox, and their associates.1'2 I would like to emphasize that all strains, regardless of how highly attenuated, retain the property of multiplying and destroying cells in the monkey spinal cord. The degree to which this property is retained, however, varies over an enormous range as we progress from the virulent strains to the highly attenuated ones suitable for vaccine use. The laboratory technics are such that different degrees of neurotropism, even among attenuated strains, can be readily detected. Thus the Lederle strains were found to be slightly more neurotropic for monkeys than the Sabin strains.1'2 The Public Health Service has approved for licensing any strain which is no more virulent than the Sabin strains, and which is at least as potent immunologically. This seems to have been a wise decision in view of the few cases which have developed followed the use of nonlicensed strains
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LIVE POLIOVIRUS VACCINE

in Miami and in Berlin. The laboratory seem to have some epidemiologic significance for indicating limits of neurovirulence which can be tolerated by man. The vaccine strains differ from the virulent strains as regards both their in vivo and their in vitro properties (see Table 1). It is well documented that the vaccine strains have produced progeny, not only in the laboratory but also in vaccinated persons, which have genetically different properties from the virus particles in the oral vaccine. This difference is one of degree, ranging from alteration in only one of the in vitro markers studied to full intracerebral virulence for the monkey. The rate of change of a viral character is determined by the number of loci within the genome which control this character. Thus a particular character, if it is controlled by a single locus, will vary as this one locus mutates. However, if it is controlled by the combined activity of a number of loci, then it will be
tests

much more stable, varying only when all relevant loci have mutated. Genetic studies on poliovirus have shown that some characters are much more stable than others. Thus the antigenic structure of a number of strains has not been observed to change, despite strong selective pressures to isolate new variants.9 On the other hand, dvirus changes to d + in a single mutational event with a frequency of one per 100,000 virus particles produced.'0 From the results obtained with virus recovered from vaccinated children, it can be said that sometimes no reversions occur, sometimes reversions occur in a single marker, and sometimes in more than one.12 Strains which revert in a number of in vitro markers show an increased degree of reversion in monkey neurovirulence,'3 as these properties form a single group of covarying characters." In a recent study,'3 85 strains recovered from children vaccinated with the Sabin strains were tested for their degree of alteration. In 35,

Table 1-Properties of Virulent Poliovirus and Attenuated Vaccine Strains*
Virulent

Attenuated

Neurovirulence: Chimpanzees (intraspinal route) Monkeys (intrathalamic route) Monkeys (intraspinal route) Monkeys (intramuscular route)
Viremia:

++

++ ++ +

+

Monkeys (intramuscular route) Man (oral route)
In vitro markers: d (bicarbonate) MS (monkey stable cell line) rct/40 or T Elution Cystine Intratypic antigenicity

++ +

-(1) +(2) -(3)* -(1) + (2) - (3)*

+

independent
stable

requirmg

stable

ii

* The data on which this table is based may be found in references 1 and 2, except for the results on viremia following infection with the vaccine strains.8 For the attenuated Sabin vaccine strains, viremia has been found only with Type 2.

MARCH. 1962

473

Table 9 In Vitro Markers and In Vivo Properties of Sabin's Type 3 Vaccine Virus and Its Progeny After Multiplication in Children
Vaccine
Vaccine

Progeny
2576

Vaccine Virus
d T (rct/40) Antigenic K value Chimpanzee paralysis (spinal injection) Monkey paralysis (thalamic injection)

S-33 (20 Days PostPostvaccination) vaccination)

Progeny

(21 Days

42 -

+ + 42* + +

+ + 42
+

-

+

Monkey paralysis (spinal injection) Monkey paralysis (muscular injection) Monkey viremia after day 1 (muscular injection)

30,000t
-

<lot
+

lot
+
+

+

* A wild virus recovered from a child in the area prior to the administration of vaccine yielded a K value of 13 when allowed to react with vaccine antiserum. t Listed are the tissue culture doses of virus necessary to paralyze 50 per cent of the monkeys wben inoculated by the intraspinal route.

there was no change in the virus, in 38 others there was a change in the d but not in the T (rct/40) marker, and in 12, changes occurred in both these characters. The Type 3 vaccine strain undergoes the greatest change, for all of the d+T+ isolates obtained were Type 3. How can one be certain that the virus recovered from a vaccinated child is derived from the vaccine and is not a wild virus circulating in the area? Most characters of poliovirus are so unstable that they cannot be used for fingerprinting purposes. The one marker now believed to be stable is the intratypic antigenic structure of the virus. McBride" has shown that the rate of inactivation of a poliovirus strain by its homologous serum is faster than that achieved by serums for other strains even though they belong to the same
474

type. The more rapid the inactivation, the larger is the K value of neutralization.
D

Vo

K=- 2.3 log

Vt where D is the dilution of serum used, t is time in minutes, VO is original virus concentration as PFU per ml, and Vt is infective virus concentration at time t. Two other tests for measuring intratypic antigenicity have been proposed. One determines the ability of virus to diffuse through agar containing antiserum to a layer of susceptible
t

cells.13 The other measures the decrease in size and number of plaques after infected monolayer cultures have been placed under agar containing antiserum.14 The latter test designed by Wecker suffers from the fact that the virus-antibody reaction is not separated
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LIVE POLIOVIRUS VACCINE

from the virus assay system and may lead to designating certain vaccinederived strains as wild strains.'5 Let us now examine the data obtained on two strains from two different localities, the first being excreted by a child in Mexico City and the second by a child in Cincinnati, each three weeks after vaccination. The data in Table 2 show that the two strains retained the K value of the vaccine and thus can be identified as true vaccine progeny. The strains regained the d+ and T+ properties associated with virulent viruses, and in chimpanzees and monkeys they act like virulent viruses both as regards their capacity to produce paralysis after inoculation by the intraspinal, intracerebral, or intramuscular routes, and by their production of viremia. One of the factors for rejection of the Lederle-

Cox and the Koprowski candidate strains by the Public Health Service was their higher degree of neurovirulence for monkeys, and yet it must be emphasized that strains excreted by Sabin-vaccinated children and their contacts are often more virulent than the rejected vaccine candidates. Fortunately this problem is lessened by the observations that vaccine viruses tend to spread to a lesser degree in certain communities than do virulent viruses. Nevertheless the occurrence of reversions should influence public health officials in their recommendations of how the vaccine should be used. We would now like to review some data obtained on strains recovered from three different epidemic areas following the use of live virus vaccines.16 These strains were kindly made available to us

Table 3-Intratypic Antigenic Studies with Type 1 and Type 3 German Strains (the Type 1 and Type 3 Vaccine Strains Used in Berlin Have Normalized K Values of 100)
Clinical
Virus Type
Area

Time of Virus Isolation

Vaccination Status

Descriptions of Persons Yielding Strains

Number
of Strains

Normalized
K Values

1

Berlin
West Germany
Berlin
Berlin

Oct., 1959May, 1960 JulyDec., 1959
JuneAug., 1960 Oct., 1960 June, 1960Apr., 1961

Pre None

healthy; polio; aseptic men.

6 6

39-56

polio healthy; polio*
polio polio
fever

43-54 92-100 65 49-62 49 92-103 38 33-38

Post
Post

9
1 6
1

West Germany
3

None Pre

Berlin Berlin Berlin West Germany

May, 1960
June-

Post Post None

healthy; polio
polio polio

6
1

July, 1960 Oct., 1960
Nov., 1960Apr., 1961

2

* Includes a strain recovered from spinal cord of a fatal case whose son had received vaccine.

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475

Table 4-Poliomyelitis in Belgrade, 1960 Number of Cases Reported
4 15 24 13 6 1 0

Month

Number of Cases Number of Polio Strains Islated Tested Type 1 Type 2 Type 3
4 10 20 10 5 0 0

January-May June July August September October November-December

4 6 10 7 2 0 0

0 1 0 0 0 0 0

0 2 3 1 1 0 0

by investigators in Germany, where in Berlin a trivalent vaccine was used in 1960; and also by investigators in Yugoslavia, where in 1960 they produced live Type 1 vaccine from the Sabin strain and used it to combat an epidemic; and lastly, by investigators in Israel, who used Type 1 vaccine (manufactured by Wyeth laboratories from the Sabin strain) to combat the 1961 polio epidemic in that country.

were recovered chiefly from polio cases,

but, in addition, one was recovered
from a case of aseptic meningitis and one from a healthy child. When tested against the antiserum (Type 1 vaccine) which is normalized to a value of 100 against the vaccine strain itself, none of these strains showed normalized values higher than 56, indicating that even though they were Type 1 strains they were not identical with the vaccine strain. Beginning in June, after the vaccine had been fed, the strains recovered from six vaccinees in Berlin had K values almost identical with that of the vaccine itself. In addition, three contacts who developed polio in July and August yielded viruses with homologous K values. It is important to know that one of these strains was actually recovered from the central nervous system of a man whose son had received the vaccine. By October, a strain recovered from a contact of a vaccinated child was now found to be different from the vaccine strain, and presumably this person was actually infected with a wild strain unrelated to that in the vaccine. In other parts of Germany, strains recovered in June, July, August, and November, 1960, and two strains recovered in 1961, were all different antigenically from the vaccine strain, even though they all belonged to polio Type 1. Other strains which were tested and found different from the vaccine strain
VOL. 52. NO. 3, A.J.P.H.

Berlin* Between May 11 and 20, 1960, trivalent vaccine was fed to 280,000 persons.17 Twenty-three cases of poliomyelitis occurred in the vaccinees, 17 of these between 9 and 21 days after feeding, and 6 at a later period. In addition, 15 cases occurred in contacts and 8 cases occurred in noncontacts. There had been virtually no clinical poliomyelitis in Berlin prior to the feeding of the vaccine strains, even though polioviruses had been recovered from some normal children as part of a survey prior to the feeding of the
vaccine.

Table 3 lists the K values normalized to 100 found for six strains recovered in Berlin in 1959 and 1960, prior to the administration of the vaccine, and also the K values of strains recovered in 1959 from West Germany. These strains
* Data kindly supplied by Dr. Henneberg, Robert Koch Institute, Berlin, Germany.

476

LIVE POLIOVIRUS VACCINE

used in Berlin were the Sabin Type 1 vaccine strain, a strain recovered from a fatal case in Houston in 1958, and a strain recovered from a paralyzed child in Israel in 1958. The bottom of Table 3 presents the data obtained on Type 3 strains from Germany. A strain obtained before the vaccine was used gave K a value of 49, different from the vaccine virus. Strains isolated in May, June, and July from healthy vaccinees all yielded K values showing that these strains were the same as the vaccine strain. A strain isolated in June from a paralyzed contact of a vaccinated child also yielded a value of 102, indicating identity with the vaccine strain. A strain isolated some months later, in October, from a nonvaccinated person who developed clinical polio, yielded a value of 38. If we compare these values with the K values of strains recovered from West Germany in November, 1960, or in April, 1961, or from Mexico in 1958, or Israel in 1960, these strains are all different from the Type 3 vaccine used.

Yugoslavia*
We now turn to a consideration of strains recovered following use of the Sabin Type 1 strain in Belgrade, Yugoslavia, in 1960. This vaccine was made in the virus laboratories of the Institute of Hygiene in Belgrade in 1960, and was promptly used in the field. Of a total population of 732,000 persons, in Belgrade, 189,000 were under the age of 16. Of the juvenile population, 71 per cent were vaccinated, beginning in mid-July and continuing through September. As shown in Table 4 the epidemic in progress was predominantly due to Type 1, even though strains belonging to the other types were also isolated. This epidemic reached its
* We are indebted to Drs. L. Stojkovic, M. V. Milovanovic, and R. Mirkovic for making the data and strains available to us.

peak earlier than usual for epidemics in this area, and its seems that the vaccine was effective in bringing the epidemic to a close sooner than expected. Strains which occurred in vaccinated individuals following the use of the vaccine were kindly made available to us for study. Antiserum against the Sabin Type 1 vaccine strain (LSc 2ab) was used and the K value again normalized to 100. Results on the Yugoslav strains are shown in Table 5. Four strains recovered in the prevaccination period, from patients paralyzed during the first part of July, 1960, yielded K values between 44 and 69, indicating that they were different from the vaccine strains. The same may be said for 11 strains isolated from patients who became ill between the eighth and 61st day after the ingestion of virus. One of the Belgrade viruses was actually isolated from the central nervous system of a fatal case who became sick 22 days after vaccination. The simplest interpretation of these data is that the vaccine was not effective in building immunity in these people and they remained susceptible to the epidemic
Table 5-Intratypic Antigenic Studies on Type 1 Yugoslav Strains, 1960 (the Sabin Type 1 Vaccine Strain Has a Normalized K Value of 100)
Strain
Pre-A* Pre-B Pre-C Pre-D

Normalized K Value
64 68 69 44 67 72 55

Strain

Normalized K Value 63 63 61 59 61 44 70 63

Post-16 Post-18 Post-21

Post-21t
Post-23 Post-25 Post-57 Post-61

Post-8t Post-9 Post-12

* Prevaccination strains from patients paralyzed during first part of July, 1960. t Post-8, onset eight days after vaccination. Samples yielding virus were usually collected within three to ten days after onset. I Post-21, virus from CNS of fatal case with onset 21 days after vaccination; patient died four days after onset.

MARCH, 1962

477

virus. One cannot tell what the reason for this might be, but one can hazard a guess that it might have been due to interference by enteroviruses which occur in the summer in temperate zones.

Israel* Epidemic poliomyelitis first made its appearance in Israel in the fall of 1949, increasing to an attack rate of 130 per 100,000 in 1950, when 1,621 cases occurred.'8 Poliomyelitis maintained itself in epidemic form through 1956, with annual attack rates never falling below 23 per 100,000. In 1957, in association with widespread use of Salk vaccine, there was a sharp decrease to 57 cases. However, a steep rise occurred in 1958 (573 cases), which stimulated the introduction of a Salk vaccine of higher potency.19 This was followed by a decline in 1959 (36 cases) and 1960 (33 cases). The sporadic development of cases observed in 1959 and 1960 continued until March, 1961, when a new outbreak began. By the end of May, 140 paralytic Type 1 cases were reported. About half had been among Arab children, this being the first time that poliomyelitis outbreaks in Israel had involved Arab villages to this extent. Where Salk vaccine had been widely accepted, a few cases had occurred, either in Arab or in Jewish children. In the epidemic areas, the attack rate among Arab children in the first two years of life was extraordinarily high, about one per 100. Two possible explanations came to mind, either or both of which may have resulted in a delay in the initial experience of young Arab children with poliovirus, so that their first exposure to virulent poliovirus came in 1961, at a stage in life when they were no longer under the protection of maternal antibodies. (1) The high proportion of
* We are indebted to Drs. Y. Yoffe, E. Eylan, and N. Gold-blum for making the preliminary results on the 1961 outbreak available.

Salk vaccinations and/or the 1958 Type 1 epidemic may have resulted in greatly curtailed circulation of Type 1 poliovirus during 1959 and 1960. (2) The sanitation and personal hygiene practices among Arabs in Israel may have improved to such a degree that poliovirus infections no longer circulate year in and year out among all children early in life. Type 1 oral vaccine at a dose of 105 3 TCD5() was used. It possessed the same low degree of monkey neurovirulence as Sabin's original seed, but was free of the contaminating vacuolating virus.20 Lots 004 and 005 supplied by the Wyeth Laboratories were parts of the same lots as those used in clinical trials in Houston, Tex., and in Cumberland, Md. Enough vaccine for 250,000 children (the total population under four and one-half years) was supplied, with each lot further subdivided by incorporating molar MgCl2, a polio vaccine stabilizer,22 in one-half of the lot. Vaccine was administered to over 90 per cent of the children in this age group in June, with most of the vaccine distributed in the first two weeks. Prior to 1961, Israel had its peak occurrence of cases in the months June, July, and August. In 1961, the number of cases occurring in June was half the number (32) which occurred in May (65), and in July the cases dropped still further (10); in August there were only four, and none have occurred since. Thus the epidemic had its peak in May, with almost no cases after July -the first time that this pattern has ever been observed in Israel. At least three explanations may account for the results: (1) The live poliovirus vaccine quickly and effectively brought the outbreak to a halt. (2) Because of the 1958 outbreak and the continued use of potent Salk vaccine since then, the number and proportion of susceptibles in the population were so low that the epidemic came to an end sooner than had occurred in the years
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LIVE POLIOVIRUS VACCINE

Table 6-Intratypic Antigenic Studies on Type 1 Israel Strains, 1961 (the Sabin Type 1 Vaccine Has a Normalized K Value of 100)

Strain
Pre-A* Pre-B Pre-C Pre-D

Normalized K Value
67 72 70 64

Strain

Normalized K Value
74 70 67 66 68

Post-3t
Post-7 Post-7 Post-10 Post-30 Post, no vaccine

59

* Prevaccination strains, May, 1961. t Post-3, paralysis three days after vaccination.

before vaccination was practiced. (3) A combination of the above, namely (a) the killed-virus vaccine was effective but did not cover everyone, and (b) the live virus vaccine further raised the level of community immunity to a height sufficient to stop the epidemic. Strains isolated from patients before and after vaccine administration were subjected to the intratypic antigenic tests. As shown in Table 6, Type 1 strains isolated before the vaccine was fed, in May, 1961, were different antigenically from the vaccine strain. The same can be said for the strains recovered from five patients three to 30 days after the vaccine was administered, and from a nonvaccinated child. It seems that, as in Yugoslavia, the postvaccination cases in Israel were caused by wild strains rather than by the vaccine strain. However, this must be a cautious interpretation, for we did not have available for study strains recovered from healthy vaccinated children in these two areas. We have been carrying out a fairly extensive analysis of a trial with the Sabin strains in Houston, but have had to interrupt the analysis of this material because of the studies involving the three outbreaks following the use of vaccines in the
MARCH. 1962

field. Recently, we have returned to the analysis of the Houston strains recovered from vaccinated children, and the results suggest that the Sabin Type 1 strain is not as stable antigenically after multiplication in children as in the Sabin Type 3 strain or the Lederle strains of all three types. If this is so, then data such as presented above for Yugoslavia and Israel cannot be used as absolute proof that the strains recovered from the vaccinated children who subsequently had paralytic polio are wild strains rather than progeny of the vaccine strain. However, more data must be obtained to elucidate this point, and the data on Sabin Type 1 strains must be looked upon as a report of work in progress.

The A Marker This discussion has been concerned with markers for identifying populations of virus particles which breed true genetically, at least under standard conditions in the laboratory. A new marker recently has been discovered2' which offers interesting potentialities in the development of attenuated strains, and also in maintaining their constancy during the course of manufacture. If virulent or attenuated strains are heated at 50° C for 15 minutes in water, one loses 3 to 4 loglo units of virus. However, in the presence of AlCl3, the atTable 7-Correlation of the A Marker with the d but not the T Marker
d+T+ d+T- d-TTotal 12 4 9 25

A+ A+ ATotal

2 0 0
2

10 4 0
14

0 0 9
9

A + = >99 per cent inactivation at 50° C in 10 mM AICl3. A- =No inactivation.

479

Table 8-Separation of Attenuated Type 2 Virus from a Mixture witl Virulent Type 1

plus typing

sera

1I
Type 1 serum

plus typing

sera

Type 2 serum

Types 1+2
sera

Type 1
serum

Type 2
serum

Types 1+2
sera

4.2x104 5.11O4

< 101

4.9x104

<101

<101

neurovirulence.1"-13

tenuated strains are rendered thermoresistant, in contrast to the virulent strains, which become even more thermolabile. This property can be used as a marker to differentiate strains: an A + strain shows at least 99 per cent inactivation when heated at 500 C in 10 mM AlC13; an A- strain is one which shows no inactivation under these conditions; and an A± strain is intermediate. Table 7 records an analysis of 25 strains which have been measured for the d and T markers,' 123 as well as for the A marker. One can see a clear correlation between the A and the d markers. As mentioned earlier in this discussion, strains possessing positive markers are those possessing positive

1 particles, respectively, were recovered in amounts equal to those originally placed in the mixture. When a mixture of Types 1 and 2 serums was added to the unheated virus, no virus was recovered, for all of it was blocked. On the other hand, when Type 1 serum was added to the heated mixture, we recovered 4.9 x 104 virus particles, all of the original attenuated Type 2 variety. The Type 2 serum did not allow any virus to grow, indicating that there was no virulent Type 1 virus left in the mixture after the heating in the presence of

AlCl3.
Table 9 shows the results of an experiment in which a mixture of attenuated and virulent Type 1 viruses were separated.21 Here we have mixed the d-T- attenuated LSc strain used in Sabin vaccine, with virulent Mahoney which is d+T+. If we heat the attenuated strain alone as shown in the left-hand part of the table-in the presence of aluminum, the virus recovered in either the heated or the unheated fraction still retains the d - T - character of the LSc strain itself. The righthand side of the table shows the results
VOL. 52. NO. 3, A.J.P.H.

Table 8 shows an application of this marker to the separation of attenuated from virulent strains.2' Virulent Type 1 and attenuated Type 2 viruses were mixed. AlCl3 was added and one-half of the mixture was heated. The unheated and heated portions were then treated with typing serums. When Type 1 serum or Type 2 serum was added to the unheated material, Type 2 or Type
480

LIVE POLIOVIRUS VACCINE

with the virulent virus; unheated, the virus is d+T+, but after heating, no virus is recovered, for virulent strains are destroyed by heat in the presence of aluminum. The mixture in the presence of the AlCl3 solution, when unheated, yields the characters d+T+ as expected, for a mixture containing equal parts of attenuated and virulent viruses will yield characters only of the dominant "plus" types. However, after heating, the virus particles recovered are d-T-, which are the characters of the attenuated strain. Thus the virulent strain was completely eliminated from the vaccine by this simple manipulation.

MgCI2 Effects To turn to the effect of another cation,
it was mentioned earlier in this report that high concentrations of MgC12 were added to the vaccine used in the Israeli trial. When magnesium ions are present at the high concentration, the vaccine may be held for at least 25 days at room temperature without showing

any inactivation.22 Even at 50° C, the virus can be stabilized by high concentrations of this cation, as well as other divalent cations. As we are discussing the fate of viral populations, it is noteworthy that this simple expedient of adding MgCl2 to stabilize poliovaccine at the same time readily removes another viral population, the so-called vacuolating or SV40 virus, which sometimes creeps into live poliovirus vaccines,23 for these viruses are present as spontaneous contaminants of monkey kidney cultures.20 Figure 1 shows an electron micrograph of a purified preparation of the vacuolating virus obtained from such monkey kidney cultures. It is a virus which is either spherically or hexagonally shaped, having a diameter of about 45 my. In the figure, one can see the capsomeres present on the surface of the virus, these being 42 in number.24 While there is no evidence that this virus is pathogenic for man, it does fall taxonomically in the papova tumor virus group,25 and it would seem the better part of wisdom to be sure that it be removed from any

Table 9-.Separation of the Attenuated Virus Population from an Attenuated-PlusVirulent Mixture
ATTENUATED LSc d-TVIRULENT MAHONEY d+ T+ 1x106 PFU/mI in 12.5 mM AICd3 MIXTURE

iX106 PFU/mI in
12.5 mM

AIC13
0.5x`106 attenuated 0.5x1o6 virulent
in 12.5 mM

AICd3
HEATED NO VIRUS

HEATED d-T-

UNHEATED d-T-

UNHEATED d+T+

HEATED

d-T-

UNHEATED d+T+

Heat treatment: 500C for 15 minutes

MARCH. 1962

481

netic characters. It was hoped that the virus populations in live polio vaccines would breed genetically true and would not revert to more virulent forms. However, from the work of the past three years we should all recognize that reversions do occur, and thus the vaccine should be used in communities to minimize any potential hazard. It seems that the surest way to initiate the use of live virus vaccine in this country would be on a mass basis so that the entire population in a community receives the vaccine virus simultaneously,
Figure 1-Vacuolating virus (SV-40) stained with phosphotungstate ions. 160,000 X magnification. Lower right, reversed print showing capsomeres in greater detail. 240,000 X magnification. Electron micrographs by L. Jordan and H. D. Mayor, Virology Department, Baylor University College of Medicine.

thus lessening the number of susceptibles who might become infected with reverted virus. At the minimum, all

vaccine that was intended for human beings. Figure 2 indicates that it is relatively easy to remove vacuolating SV40 virus, since different virus populations react differently to the presence of MgCl2.23 In water, vacuolating virus is relatively stable to heating at 50° for 60 minutes, but poliovirus in water is unstable, and after this treatment can hardly be detected. However, in the presence of MgCl2, one finds a reversal of these effects; poliovirus is stabilized by MgCl2 and the vacuolating virus is quickly inactivated. Thus we have here a very useful tool, one which in the process of stabilizing the vaccine virus so that it does not have to be stored frozen eliminates contaminating viruses from the vaccine.

w

a

C)

z
U1)

w
C-)

z

w

a-

VACUOLATING VIRUS IN Mg Cl2

5

15

30

60

MINUTES AT 500C

Concluding Remarks
I have discussed ways in which virologists handle viruses, for-whatever our manipulations-we are always treating populations, usually of mixed ge482

Figure 2-Vacuolating and poliovirus (attenuated vaccine Type 1) mixed to contain 106 and 107 PFU/ml, respectively. Mixed virus suspension diluted with equal volume of (a) 2 M Mg++, (b) distilled H20. Detection of surviving poliovirus was made by plating on rhesus monkey kidney cells, and counting PFU developing in two to three days. Vacuolating virus was detected by neutralizing poliovirus with a pool of Type 1 polio antiserums (free of vacuolating virus antibody), and plating on African green monkey kidney cells. Vacuolating virus plaques were read between the 11th and 14th days.28
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LIVE POLIOVIRUS VACCINE

members of a family should be vaccinated simultaneously. Community vaccination should be done in a period of the year when the prevalence of enteroviruses is at a minimum, to avoid blocking of vaccine takes by viral interference.2'26 Actually, rapid mass vaccination was the way the large Russian field trials were conducted, and the Russians are continuing to use the vaccine in this way, with the stated purpose of minimizing potential hazards which might sometimes result from an unfavorable genetic reversion.27 By application of the newer findings concerning the reactions of cations with viruses, it is now possible to eliminate undesirable adventitious viral populations from live poliovirus vaccine and at the same time stabilize the vaccine so that it can be stored without freezing and used for weeks in the field without refrigeration. Vaccine stabilized by cations has proved to be fully potent as an immunizing agent.
REFERENCES
1. Poliomyelitis. Papers and Discussions. Fourth and Fifth International Poliomyelitis Conferences. Philadelphia, Pa.: Lippincott 1958 and 1961. 2. First and Second International Conferences on Live Poliovirus Vaccines. Pan Am. Health Organ. Sc. Publ. No. 44 and 50, 1959 and 1960. 3. Koprowski, H.; Jervis, G. A.; and Norton, T. W. Immune Responses in Human Volunteers Upon Oral Administration of a Rodent-Adapted Strain of Poliomyelitis Virus. Am. J. Hyg. 55:108-126, 1952. 4. Enders, J. F.; Weller, T. H.; and Robbins, F. C. Alteration in Pathogenicity for Monkeys of Brunhilde Strain of Poliomyelitis Virus Following Cultivation in Human Tissues. Fed. Proc. 11:467, 1952. 5. Sabin, A. B. Properties and Behavior of Orally Administered Attenuated Poliovirus Vaccine. J.A.M.A. 164:1216-1223, 1957. 6. Li, C. P.; Schaeffer, M.; and Nelson, D. B. Experimentally Produced Variants of Poliomyelitis Virus Combining In Vivo and In Vitro Techniques. Ann. New York Acad. Sc. 61:902-910, 1955. 7. Melnick, J. L. Problems Associated with the Use of Live Poliovirus Vaccine. A.J.P.H. 50:1013-1031, 1960. 8. Melnick, J. L.; Proctor, R.; Ocampo, A. R.; Diwan,

9. 10. 11.
12.

13.

14.

15. 16. 17.

18.
19.

20. 21. 22. 23.

A. R.; and Ben-Porath, E. Viremia After Administration of Live Poliovirus Vaccine. Bact. Proc. 4:145, 1961. McBride, W. D. Antigenic Analysis of Polioviruses by Kinetic Studies of Serum Neutralization. Virology 7:45-58, 1959. Dulbecco, R., and Vogt, M. Study of the Mutability of d Lines of Polioviruses. Ibid. 5:220-235, 1958. Dulbecco, R. Discussion in Second International Conference on Live Poliovirus Vaccines. Pan Am. Health Organ. Sc. Publ. No. 50, 1960, pp. 47-49. Benyesh-Melnick, M., and Melnick, J. L. The Use of In Vitro Markers and Monkey Neurovirulence Tests to Follow Genetic Changes in Attenuated Poliovirus Multiplying in the Human Alimentary Tract. Ibid., 1959, pp. 179-202. Melnick, J. L., and Benyesh-Melnick, M. Problems Associated with Live Poliovirus Vaccine and Its Progeny After Multiplication in Man. Ibid., 1960, pp. 12-30. Wecker, E. A Simple Test for Serodifferentiation of Poliovirus Strains Within the Same Type. Virology 10:376-379, 1960. McBride, W. D. The Antigenic Differentiation of Polioviruses. (Unpublished.) Melnick, J. L.; Ozaki, Y.; Diwan, A. R.; and McBride, W. D. (Unpublished.) Henneberg, G. Discussion. Fifth International Poliomyelitis Conference. Philadelphia, Pa.: Lippincott, 1961, p. 211. Yekutiel, P.; Levinger, E. L.; Muhsam, H. V.; and Yekutiel, M. Poliomyelitis Outbreak in Israel, 1950-1. Bull. World Health Organ. 12:651-676, 1955. Davies, A. M.; Marberg, K.; Goldblum, N.; Levine, S.; and Yekutiel, P. Epidemiology of Poliomyelitis in Israel, 1952-59. With Evaluation of Salk Vaccination During a Three-Year Period. Ibid. 23:53-72, 1960. Sweet, B. H., and Hilleman, M. The Vacuolating Virus SV40. Proc. Soc. Exper. Biol. & Med. 105:420427, 1960. Wallis, C., and Melnick, J. L. (Unpublished.) . Stabilization of Poliovirus by Cations. Texas Rep. Biol. & Med. 19:683-700, 1961. . Cationic Inactivation of Vacuolating Virus (SV40) in Poliovirus Suspensions. Ibid.

19:701-705, 1961.
24. Mayor, H. D., and Jordan, L. E. (Unpublished.) 25. Melnick, J. L. Papova Virus Group. Science

(In

26. Benyesh-Melnick, M.; Melnick, J. L.; and RamosAlvarez, M. Poliomyelitis Infection Rate Among Mexican Children Fed Attenuated Poliovirus Vaccines. Live Poliovirus Vaccines. Pan Am. Health Organ. Sc. Publ. No. 44, 1959, pp. 272-285. 27. Chumakov, M. P.; Voroshilova, M. K.; Drozdov, S. G.; Dzagurov, S. G.; Lashkevich, V. A.; Mironova, L. L.; Ralph, N. M.; Gagarina, A. V.; Ashmarina, E. E.; Shirman, G. A.; Fleer, G. P.; Tolskays, E. A.; Sokolova, I. S.; Elbert, L. B.; and Sinyak, K. M. Some Results of the Work on Mass Immunization in the Soviet Union With Live Poliovirus Vaccine Prepared From Sabin Strains.

press.)

Bull. World Health Organ. 25:79-91, 1961. 28. Stinebaugh, Sara, and Melnick, Joseph L. Plaque Formation by Vacuolating Virus, SV40. Virology

(In press.)

Dr. Melnick is professor of virology and epidemiology, Department of Virology and Epidemiology, Baylor University College of Medicine, Houston, Tex. This paper was presented before the Epidemiology Section of the American Public Health Association at the Eighty-Ninth Annual Meeting in Detroit, Mich., November 13, 1961. This study was aided by a grant from the National Foundation.

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