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Adaptation of the 17D yellow fever virus to mouse brain by serial passage
445 'FRANSACTIONS (~F THE ROYAL ~OCIET~t Oi,
TROPICAl, ~%IEDICINE AND t Iv(;IFNE. Vol. 53. No. 6. November, 1059.
A I ) A P T A T I O N OF T H E 17D Y E L L O W FE\rER VIRUS T O MOUSE BRAIN BY SERIAI, PASSAGI" BV
('aptain 1'. 1). I\It'2ERS, Royal Army ~,lcdic;fi Corps *
i From the Federal Laboratory ?~'ervice, Headqztart~* ~, ~,'aba, Logos, X&eri,:~)
T h e p r o d u c t i o n at Yaba, Nigeria, of a 17D yellow fever m o u s e - b r a i n vaccine, for use in h u m a n i m m u n i z a t i o n by scarification, p r o m p t e d an e x p e r i m e n t designed to investigate the b e h a v i o u r of the 17D virus on serial passage in m o u s e brain. It was t h o u g h t that this m i g h t lead to a strain of yellm~ fever virus which, while still c o n f o r m i n g to the W H O req u i r e m e n t s for 17D c h i c k - e m b r y o seed vaccine (and being, therefore, in all p r o b a b i l i t y safe for use as an i m m u n i z i n g agent), would yield a higher titre in m o u s e brain, giving a cheaper vaccine. At the same time it was felt that such a strain m i g h t also p r o d u c e a significantly greater c o n v e r s i o n rate a m o n g s t those vaccinated t h a n that p r o d u c e d bv 17D chicke m b r y o or the first passage 17D m o u s e - b r a i n vaccine in use at present. T h i s work is r e p o r t e d in two parts, the first dealing with the changes i n d u c e d in the 17D virus as d e m o n s t r a t e d in mice, the second dealing with the changes d e m o n s t r a t e d in I n d i a n rhesus m o n k e y s ( 3 t a c a c a mulatta).
1. TIlE CHANGES IN THF VIRUS OBSERVED IN MICE MATERIAI.S AND METHODS Notation The term " 17D-MA " (the letters MA standing for mouse adapted) refers to the 17D vellmv fever virus grown in mouse brain. As a suffix to this is added the passage lexel, rims 17D-'AL~-I refers to virus after one mouse-brain passage, that is, material harvested from mice suffering a specific 17I) yellow fever encephalitis as a result of the intracerebral inoculation of 17D chick-embryo grown virus. 17D-.\IA-2 is material harvested from mice whep paralyscd following the inoculation of 17D-MA-t material, and so on.
Bur/Fred normal saline ( BI :\',5') This was prepared by adding sulticient sodmm chloride to produce a concentration of 0 85 per cent. in Sorensen's M15 phosphate buffer solution, pH 7.4. It was sterilized in the autoclave. 0.75 per cent. Bovine albnmin saline (B,4/S) A stock solution was prepared by dissolving Armour brand bovine albumin (fraction V) in B/NS to make a 7.5 per cent. solution, which was filtered throut~h a seitz pad. For use, this was diluted to 0.75 per cent. with further B / N S . * Thanks are due to Sir Samuel Manuwa, Chief Medical Adviser to the Federal Government of Nigeria, for permission to publish this paper. I am grateful to Dr. F. N. Macnamara, Director, West African Council for Medical Research, who has given a great deal of advice. Mr. R. C. Kent. Messrs. l,. D. Jaja, and H. Imemogha rave techMcal assistance.
44(~
\ I ) A I ' T A T I O N OF 171) YELLOW FEVER VIRUS "10 MOUSE BRAIN
Mice Mice were derived from the nlousc colony attached to the Yellow Fever Vaccine Laboratory. T h e colony is of Swiss albino mice, obtained from the Virus Research Institute, Entebbe, Uganda. T h e standard " group " of mice numbers six individuals, taken at random from those of the age-group concerned.
The viruses T h e 17D yellow fever virus employed was derived from vaccine lot 1982 nlade m the Rockefeller Laboratories, New York. From this was prepared in chick embryos a primary seed lot (PSi) which has been in use for the preparation of secondary seed lots for vaccine production in this laboratory, and which was the starting point for two of the passage series reported. T h e third passage series was initiated with secondary seed lot S2/I)S.,, prepared in chick embryos inoculated with material from primary seed lot PS,,. T h e Dakar vaccine strain of yellow fever virus was derived from I)akar vaccine batch A98, alnpoules of which were obtained from the West African Council for Medical Research. This was used as it was, or, as the titre was low due to prolonged storage, after one further passage in mice.
Passage details Of the three passage series, the two initiated with the PS 2 virus were designated PS., 1 and PS,,/2 respectively. T h e third series, with the S2/PS., starting point, was called the S2/PS 2 series. Twelve groups of mice were used at each passage level in the I)S,j1 and PSi/2 series, and six at each level of the S2/t S~ series. T o initiate the passages, the contents of an ampoule of the appropriate virus was rehydrated with B/NS., and inoculated in 0.03 ml. amounts intracerebrally into mice 35 days old. T h e first mice to become paralysed as a result of yellow fever encephalitis were separated, chloroformed, and the brains removed aseptically. T h e brains (numbering between three and six--these figures being adhered to throughout the passages) were placed in a sterile mortar with a little powdered neutral glass, and ground, adding slowly 3 ml. of BA/S per brain. When a fine emulsion had been produced, it was poured into a sterile universal container, and stood in a refrigerator (plus 4°C.) for 1 hour. One ml, of the supernate was then removed, and diluted to 1:10 by the addition of 9 ml. BA/S. This approximate 1:100 dilution of mouse brain material was used as the inoculum for the next group of mice in the series, and, where applicable, as the 1 0 for titration. This procedure was used unchanged for all the passages in all three series, except that in the PS._,/2 series some of the supcrnate before dilution to 1:100 was ampouled in {I. 5 ml. amounts, and freeze-dried, for subsequent reference. Titration of the 1:100 passage material was carried ont at each level in both the PS2/1 and PS2/2 series. In this fashion, the PS.j1 and $2,'1 S 2 series were continued for 28 consecutive passages each, and the PS,,/2 series for five passages. Care was taken to maintain accurate records of the individual mice, the mouse record cards being marked at the same time each day. T h e mice were observed for 21 days, those falling paralysed first being the ones used for passage purposes. In calculating the axerage survival time (A.S.T.) these mice were considered to have died on the day on which they were used for passage. Any mouse falling sick more than 2 days before the retnainder in tile group, was discounted for A.S.T. calculation. T h e amount of virus contained in the 1:100 mouse brain passage material was such that 100 per cent. of mice inoculated with it, (tied. T h e age of the mice used was nearly ahvays 35 days on the (lay of inoculation. If
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insufficient 35-day-old mice were available on a given day, tile deficiency was made up with mice 34 or 36 days old. The limits of age used were 32 to 38 days. Not less than {t() per cent. of the mice were, however, 35 days old exactly when inoculated.
Titrations Tenfold dilutions of the material to be titrated were prepared in BA/S. These were inoculated in 0.03 ml. amounts into not less than one group of 35-day-old mice per dilution. The 50 per cent. end point was calculated accordin~ to the method of REED and MUEN('II (1938).
The reference levels As it was obviously impracticable t{) investigate exhaustively the character of each of the viruses obtained at each passage level, certain " reference levels " had to be chosen. These were the 17D-MA-1, 17D-MA-3 and 17D-MA-17 viruses. Chick-embryo grown 17D virus and the strain of French neurotropic yellow fever virus used in the preparation of the Dakar scarification vaccine were added for comparison, as " controls."
The preparation of the reference level materials 17D-MA-1. :\ series of batches of 17I) mouse-brain vaccine produced routinely by the laboratory for human immunization by scarification were used. This vaccine is identical with 17D-MA-1 material, being produced by a development of the second method described by CANNON and DEWI:IURST (1955), from the brains of mice inoculated with 17D chickembryo seed virus.
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ADAPTATION
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Fig. 1, The changes observed in the Average Survival Time of adult mice m three serial passage experimems with the 171) yellow fever virus.
Average survival time calcu&tion T h e A.S.T. of 35-day-old mice following intracerebral inoculation with the various viruses under consideration was investigated in two distinct ways. Firstly, as the passages proceeded, the average time between inoculation and death was calculated at each passage level for each of the three passage experiments. Secondly, to allow for the effect of virus dose on A.S.T., each of the reference level viruses, and the two " control " viruses, were subjected to a series of t:~trations (10 for the Dakar vaccine strain, 20 each for 17D-MA-3 and 17, and 30---not done specially, but extracted from vaccine production records- for 17D-MA-1 and 17I) chick-embryo vaccines), l ' r o m each titration, the A.S.T. at each dilution was calculated, and the results from each of the separate materials grouped according to the log of the \irus dose, to the nearest whole n u m b e r (Table). In this connexion, only mice dying of a characteristic yellow fever encephalitis were included in the calculations. This is contrary to usual practice, which has been to allot some arbitrary figure for survival time to those mice remaining alive at the end of the experimental period, and including this ']'ABLE. The average survival time of 35-day-old mice inoculated intracerchrally with five different strair, s of yellow fever virus, related to virus dose. Average surx ival time in days, with : Log virus dos(,
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figure in A.S.T. calculations. Justification for the change is found in the clear-cut picture obtained in Figure 2. It will be seen that the final average of the 10, 20 or 30 observations at each of the logarithmic steps of virus dosage is obtained (at all but the highest dilutions) f:rom n o t less than 60 individual mice (for the I)akar strain) or 120 or 180 for tl~e r~thers.
Reactions of immature mice As an extension of the above, it was decided to discover tile mortality ratios of immature mice inoculated extraneurally with varying dosages of the various rd'erence level viruses and controls. For the purposes of the experiment, two age-groups were selected " younlz mice " (13-15 days old), and " infant mice " (3-5 days old). The procedure ;~as to prepare serial tenfold dilutions of the material under test in I{A S., each dilution being inoculated intracerebrally into one group of adult (35-day) mice for titration of tile agent. Next, the dilutions x~hich it was desired to test were inoctflated into immature mice (0.f16 ml. intraperitoneaily into young mice, and (I./13 ml. subctEtancouslv into infant mice), 10 groups of young mice, and four groups of infant mice c,sually being e,,nployed at each dilution, though the exact number varied with thc availability of mice of ti~,: correct age. The mice were observed daily for 21 days, individual records being kept. This procedure was repeated as required with all the virus materials, with the excepti(,~ (~f t71)-5IA-3, which was not lcsted in i m m a t u r e m i c e . RESUI:]'S
lver~w survival times Figure I illustrates graphically the changes in A.S.T. in each of the three passage experiments. The first two points in the S,,,:PS,, series arc omitted, as, due to insufficient mice, only three groups were used for each of the first two passages of this series. Included il~ the lower part of l:igurc 1 are the values for the titrcs of the passage materials observed in the PS.~/1 and PS2/2 series. The table gives the results of the A.S.T. calculations for the reference level and " control " viruses, related to virus dose as described above. Fi~zure
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2 expresses these graphically by plotting the 1o~ of the virus dose against the reciprocal of the A.S.T. in days. Mortality ratios in inzmatu;'e mice Figures 3 and 4 show graphically the mortality ratios in young and infant mice (as defined above), respectively, subjected to varying dosages of the reference and control viruses. These results are not given in greater detail, as, contrary to expectations, they proved to be of little value in the present context. MORTALITY
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THE CHAN{;ES IN TIlE VIRUS OBSERVE[) IN RHESUS MONKEYS (]Flacaca mu/atta) MATERIALS AND METHODS
Materials and methods dcscribed in section 1 of this paper should be referred to fm~ details not included here.
Monkeys All the monkeys used were rhesus monkeys (31. mulatta) received from India in the same consignment, 6 months before the experiments recorded here were begun. At the time they were used, they were apparently fit, and their average weight x\as 4!~ lb. (2. {) kg.), with limits between 3~ and 5½ lb. (1.7 to 2.5 kg.).
Inoculation and subsequent handlin;: The animals were separated into individual cages 7 days beforc their inoculation. From this time on, they were examined, weighed, and had their rectal temperatures recorded daily. On the day of inoculation, the monkeys were bled from the femoral vein to obtain serum for a pre-inoculation mouse-protection test, and the scalp was shaved. They were then anaesthetized with nembutal administered intravenously, and the skin and skull over the left frontal lobe perforated after the application of spirit by means of a dental burr held in the handpiece of a dental machine. The inoculum (which was reconstituted a;ld diluted in B/NS. to give an approximate dose per 0.25 ml. of 10,000 adult mouse IA)a ), was kept on ice, and was introduced by means of a tuberculin syringe, 0.25 mh being irmculated per monkey, at a depth of one inch (2.5 cm.). At the completion of the inoculations, the inoculum was titrated in mice 35 days old, two groups of mice beinf_, employed per tenfold dilution. The monkeys were bled on the 2nd, 4th and 6th days following inoculation for demonstration of circulating virus. The blood was allowed to clot, and was lightly centrifuged to obtain serum. This was inoculated intracerebrally undihtted, and diluted 1:10 and 1:100 in B/NS., into not less than one group of 35-day-old mice per dilution, 0./)3 ml. being inoculated per mouse. The mice were observed for 21 days, deaths from characteristic yellow fever encephalitis being recorded. Scrum virus titres were calculated by thc mcthod of REED and MUENCH (1938). The experimental period terminated on the 30th day after inoculation, when the surviving monkeys were bled to obtain serum for a post-inoculation protection test, and returned to the main pens. Those monkeys which, on their daily inspections, showed any change in bcha~iotlr, or appeared weak, were singled out for special attention. Daily records of their clinical condition were maintained, and, if and when they became so sick as to appear moribund, they were killed. The brain, and in some instances, the spinal cord, were removed. Duplicate contiguous pieces of the cerebrum, thalamic nuclei, cerebellum, pons, and, sometimes, cervical and lumbar cord (the samples from the brain being taken from the side remote from the site of inoculation) were collected. One of each pair was placed in fixative for histological examination, the other in a sterile container, and stored at minus 65~C., for virus titration. After removing the brain, each monkev was submitted to full postmortem examination. At a convenient time, the pieces of brain for virus titration were thawed, and emulsified by grinding with powdered glass, adding B,:NS. to make a 1:2 dilution of brain material. The
452
AI)APTATION OF ] 7 i ) YELLO\~" I,'I,]VER VIllUS 1'(7 MOUSE B R \ I \
resultant suspension was stood in the refrigerator for 10 minutes, the supernate being designated 10 °, titrations being carried o u t in tenfold dihltions, onc group of 35-day-old )nice being employed at each dilution. Twenty-one monkeys were available at the commencement of the expcrhnental period. Of these, six were used to test the 17D-MA-17 virus, six for the 17D-MA-1, six for 17I) chick-embryo vaccine, and the remaining three for the 17I)-MA-3 virus. Of these, one of the six inoculated with 171)-MA-I material died immediately following inoculation.
Protection tests T h e pre- and post-inoculation serum samples from each of the monkeys which survived the experimental period were paired, and with the pre-inoculation sample from those that were killed, were subjected to protection testing. T h e test used was the SMvrHm:R:'< (1945) intraperitoneal " 1 per cent. virus " test in immature mice. Each serum sample was put up undiluted, and at a dilution of 1:4 in I}A/S.
RESULTS
Virus dose Titration of the inocula employed gave the following rcsuhs, expressed in i.1)-.,)inoculated per monkey : iA)su 17D chick-embryo material 12,08(i 17D-MA-I 8,53~) 17D-MA-3 16,6()() 17D-MA-17 5,90() (~h'eulating ,:irus All the monkeys showed circulating virus ,m one (;r more of the three occasions on which each was tested. T h e level of circulating virus was nearly ahvays 1.w the commonest positive finding bcing a " trace," that is, less than one LD~) per 0.03 ml. of undiluted monkey serum. T h e highest titre fo,,md was one of 60 Ll)a0 per 0.03 ml. of serum. No pattern could be deduced from the rcsults, and there appeared to be no relationship betx~een the amount of circulating virus demonstrated, and 1he strain or titre of the inocuh]m.
Accepting as the upper limit of normal for tile rectal temperature of rhesu:; monkeys a figure of 104 <~F. (4().0"C.), all the monkeys inoculated showed a febrile response. T h e fever lasted for between 3 and 6 days, the lowest maximum temperature recorded durh~g the febrile period being 105.0°F. (40.5%:.), the highest 106.6°I r. (41.4°C.). No relationship u as observed between the duration or height of fever and the virus inoculated, though those animals x~.hich suffered a severe cncephalitis generally had a brief period of fever, followed. at or just after the onset of encephalitic symptoms, by a dramatic fall in temperature of between 2°-7:~I". (1.1-3.9°C.) over a period of 1 or 2 days. T h e more severe the signs, the lower the temperature ; those monkeys with a mild encephalitis ha\ing a smaller drop, the temperature rising agaiu to between 103 and 1()4 F. (39.4-4().():C.) as recovery commenced. The only wav in which the fever occurring in the monkeys could be linked with the virus
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causing it was that there was a variation in the average time of onset of fever in relation t() the virus inoculated, thus " Average time between inoculation and onset ,)t t'e',cr, in days I) ,'q
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Encephalitis T h e onset of encephalitis was in exerv case but one marked by increasing drowsiness (in the lone case it was marked by tremor), which was followed in some 24 hours by increasing weakness, occasionally accompanied by minor convulsions. T h e weakness usuallv started on one side of the body, spreading, in the more severe cases, to the other. .\s the weaknc:;s increased, the animal lapsed more frequently into semi-coma, the final state being a deeply comatose, flaccid animal, which was, at this stage, killed. In those monkeys which recovered there was always a sufficient degree of hemi- or quadri-paresis accompanied by severe drowsiness or semi-coma to enable ~l confident diagnosis to be made. T h e period between tl~e onset of the s y m p t o m s and the arrival at an apparently moribund state varied between 3 and 5 days. In those animals which wcre not killed, recovery took place in between 5 and 2() days, though one was left with a residual right sided hemiparesis. As there was such a wide x ariation in the incidence of encephalitis between animals inoculated with the various strains under considcration, they will be dealt with separately. T h e incidence is expressed graphically as part of Figure 5.
454
ADkPTATION
OF
17D
YELI,OV~ FEVER VIRUS TO MOUSE BRAIN
17D chick-embryo material
No cases of encephalitis were observed in the six monkeys inoculated with this material. To this result may be added the results of routine safety tests on batches of 17I) chickembryo seed virus. The records of two such tests (all that could be located in detail) revealed that 12 monkeys had been employed, all without evidence of encephalitis. As all these tests were on the seed virus PS., or on secondary seed lots prepared from it, it is apparent that the strain of chick-embryo grown 17D yellow fever virus in use at Yaba is, to the extent to which it has been tested, non-encephalitogenic no cases of encephalitis having been recorded in 18 monkevs which have been employed in safety testing it.
l 7D-MA- 1 One of the six monkeys inoculated with this material, died immediately after inoculatiom Of the remaining five, one developed mild encephalitic symptoms on the 8th post-inoculation day, returning to apparent normality by the 13th. The results obtained from the two monkeys inoculated with the 17D-MA-1 virus referred to by CAXXO,N"and DEWHURST (1955), (during the original safety testing of 17D mouse-brain vaccine), neither of which developed encephalitis, may be included here. Thus the 17D-MA-1 material produced one case of encephalitis in seven monkeys inoculated, an incidence of 14 per cent. "7 "~ 1/D-MA-.~
Only three monkeys were inoculated with this material. Of these, tx\o developed encephalitis, both on the 7th post-inoculation day. One of these was killed on the l lth post-inoculation day to confirm the aetiology of the condition, although it is probable that the animal would have survived. The other had returned to normal, thongh with a residual right-sided weakness, by the 26th post-inoculation day. The incidence of encephalitis with the 17D-MA-3 virus was, therefore, 67 per ccnt.
17D-MA-17 Of the six monkeys inoculated with this material, all (100 per cent.) developed encephalitis between the 6th and 9th days following inoculation. Onlv one was not killed : it had returned to normal 11 davs later. Histological exami~tation I am indebted to Dr. W. G. C. Bearcroft, West African Council for Medical Research, who very kindly carried out the histological examinations. Specimens taken from all the monkeys showed lesions typical of a viral enccphalo-myelitis. The lesions were present predominantly in the cord and hrain stem, to a lesser extent in the cerebrum : thev were absent from the cerebellum. Uirus iso&tion Virus was isolated from the brains of all six monkeys killed. In nearly every case, most virus was found in the tbalamic area. The impression was gained that the amount (ff virus fell rapidly as death approached the highest titres (up to 23,(t00 LDa~) per (}.03 ml. of brain material prepared as described) being found in the thalami of animals killed in the earlier stages of their encephalitis. There was no good correlation between the severity
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of damage seen histologically, and the virus titre. The cerebellum, in most cases, was found to contain fair amounts of virus, although no histological lesions could be demonstrated. Protection tests
All frst (pre-inoculation) samples of serum were found to gi~e a negative (non-protective) protection test result. All the samples collected on the 30th day gave a positive (protectiw:) result undiluted, and all but one (which gave an inconclusive result) were positive at a dilution of 1:4.
I)ISC[-SSION
The safety of a 17D velh)w fever vaccine produced ira mouse brain has been questioned on three counts. These are, firstly, the introduction by vaccination, in addition to the 17D virus, of pathogenic organisms, particularly viruses, which might have been present unsuspected in the brains of the mice from which the xaccine was prepared. Secondly, the production of a~l allergic demyelinating encephalitis due to the iutroduction of foreign neural tissue. Thirdly, and most important, the risk of a dangerous increase in neurotropism of the immunizing virus, with the attendant danger of causing encephalitis. 'Fhesc criticisms have been discussed very briefly before (MEt.:I~s, 1957) it is with a more detailed examination of one of them, that dealing with increased neurotropism, that we are concerned here. Two criteria generally applicable to the adaptation of a virus to a new host are, a rising titre of the infective agent, and a shortening of the incubation period, with succeeding passages, until adaptation is complete. The adaptation of the 17I) yellow fever virus to mouse brain is no exception to this, a marked early rise in titre of the passage lnaterial being seen in Figure 1. That there is a true reduction in ineubatio)l period (measured in this instance by the aw~rage survival time) cannot be deduced from Figure 1, as the rising titre of itself would produce a shortening of the A . S . T . , but is clearly dclnonstrated when the A.S.T. is related to the titre of the im)culum, as in I-'igurc 2. "1'he rapidity with which both the criteria of adaptation change in the first five or six passages is most striking, and ser\es t() underline once again the considerable lability of the 17D virt,s. That these observatio)~s are not the result of a freak mutation is x;cll shown in 1,'igure 1, where the similarity of response in the three passage experiments can be seen. It would appear from this that the course of events in the adaptation of the 17D virus t() mouse brain is constant, and reproduciblc at will. The hoped-for clear-cut " spectrum " ()f mortality ratios in immature )nick, increasing the further the passages got away from the 17D chick-embryo virus, and firmllv arriving at the high mortality expected with the i)akar vaccine strain, did n()t occur, 'l'}~e Dakar virus lived up to expectations, killi,ag 100 per cent. of infant mice at very loxx dosage levels (Fig. 4), and producing (l:ig. 3) a sp.,,ooth, steadily rising curve of mortalities with increasing virus dosage, in young mice. F(~r all the other viruses examined in young mice, however, the results obtained showed widely differing mortality ratios with similar dosages of the same agent on different occasions and, apart from noting that all the strains tested were, in generai, less pathogenic than the Dakar strain, no other deduction is possible. With infant mice, there was a fairly well marked difference between 17D chick-embryo and 17D mouse-brain vaccines on the one hand, and 17D-MA-17 on the other, in that the latter approached closer to the observed mortality ratio f(,r the Dakar strain. In passing it is of interest to note that
456
ADAPTATION Ol,' I 71) YELLOD, FEVER VIii.US '['O MOUSE BRAIN
the unmodified 17D virus is capable of producing mortality in immature mice on extraneural inoculation, contrary to the generally held opinion. Before the safety tests in rhesus monkeys were started, the production of a \ accine from the mouse adapted virus at a passage level higher than the first (that is, the cxisting 17I) mouse-brain vaccine in use at present) appeared attractive. Not only had the titre risen, making the hoped-for cheaper xaccinc a possibility, but also the virus had become more pathogenic to mice, giving rise to the hope that a greater conversion rate among those vaccinated might result. The metlnod by which the safety of a given yellow few'r immunizing agent may be investigated is well known, and is routine practice in several laboratories throughout the world. The assessment of the results obtained, ho~xever, rests entirely on the very few reports of accidents involving the appearance of recognized cases of encephalitis following vaccination in man, and relating this to the bchaviour of the causal virus in safety tests in rhesus monkeys. The safety of a given strain of yellow fever virus intended for human immunization is, therefore, judged on standards which are somewhat arbitrary. In the present instance, as the criterion of safety, the U.N.R.R.A. regulations (as subsequently adopted by WHO) for 171) chick-embryo seed vaccine testing in rhesus monkeys were accepted. These (WHO, 1956) lay down limits for circulating virus (as an index of viscerotropism), production of immunity, ai~d incidence of encephalitis (as an index of neurotropism), in rhesus monkevs. Not less than six lnonkevs arc required to be used for each test. More recently, a new set of draft regulations has been published (WIIO, 1957), which proposes the increase of the minimum nulnber of mo.'nkcvs to be used, to 10 per test. In either case, the requirements as to circulating virus and the production of immunity are met by all the strains of virus tested here, and it is only the neurotropism of the various strains which is of interest. The incidence of encephalitis permitted by the regulations still in force is 33 per cent.that is, in two monkeys out of six. 'l'he draft regulations reduce this figure to 20 per cent.two out of 10. The 17D-MA-1 virus, with an observed incidence of encephalitis of 14 per cent. (one out of seven) is well inside both these limits. Thus, although the total number of monkeys tested is still small, the 171) mouse-brain vaccine fulfills all the criteria of safety for a 17D chick-embryo seed virus. All the other viruses tested, however, exceed very markedly the upper limit of what is considered safe, and thus any hope of using the mouse adapted 17D virus for human imnmnization bey(rod its first passage in mouse brain is dashed. The increasing incidence of encephalitis in rhesus monkeys as adaptation to mice of the 17I) yellow fever xirus proceeds, reflects closely the changes observed in the average survival time in mice recorded in the first part of this paper. Thus the early rapid change noted in mice is accurately mirrored in monkeys (Fig. 5), both as regards the incidence of encephalitis, and the length of the incubation period to the onset of fever. It would be interesting to know if the various 17D seed viruses in use throughout the world for the production of yellow fever vaccine (which are known to vary in encephalitogenic properties as demonstrated in rhesus monkeys) could, if tested in one centre, be placed in order on a scale of increasing neurotropism, and if mouse A.S.T.'s could be similarly graded, as has been done here, producing a picture like that seen in Figure 2, on a smallcr scale. If this were done, the " best " 17D substrain could then be chosen for use by all vaccine producing laboratories. It is a pity that the number of monkeys available for this work was so limited. The incidence of encephalitis with the Dakar vaccine strain of virus could not be demonstrated, and, although work on this aspect of the French neurotropic virus has been done (LLOYD
I'. i), MEt:,It>
457
and Iq.;NNa, 1933) there is, potentially at least, a difference between the virus tested by them and the strain of virus used at present for vaccine production at Dakar. It would also have been preferable to have used ,'note moJ~kevs to test each of the existing " refcrcnce level " viruses, particularly i 7 D - M A - 3 , :rod to have inserted more such reference strains, say at 17D-MA-2, and at the 5th and t0th passage levels, i-lox\ever, the fact that the results fit so xvcll into a smooth pattern (l:ig. 5), which agree closely with the rcstdts ~btained in mice, suggests very str,.mgly that the recorded results are not too far from those which would have been observed had more experimental animals bccn available. Although the work reported failed in its primary objcctive of providing a 1~c~ substrain of the 17I) virus, safe (according to W I t ( ) requirements) for h u m a n immunization, it has thrmvn considerably ~>orc light OI'i the safety ) f first passage 17D mo,dsc-braira vaccine as produced at prcscr~t for scratch vaccinatitm, it has als~,~ demonstrated ,~/c<: avain the considerahle lability of the " fixed " 17D strai,~ {,1: xcllow fever virus. ~I:MMARY T h e course of events in the adaptation t~f the 171) vclhm fever ~irus to mouse brain is described. T h e changes in its character as demonstrated in Swiss albino mice, and in rhesus monkeys, are recorded and compared with the bchaviour of 17D chick-embryo grown virus, and the I)akar vaccine strain of the l:rench neurotropic virus. T h e experiments showed that the neurotropic character of the virus underwent a marked, rapid change. After 17 passages in mouse brain its neurotropism had increased sufficiently to cause an incidence ~Jf encephalitis of l(}l) per cent. in rhesus monkeys inoculated with it. P,EI:ERENCES CANNON, D. A. & I)EWHUt
TROPICAl, ~%IEDICINE AND t Iv(;IFNE. Vol. 53. No. 6. November, 1059.
A I ) A P T A T I O N OF T H E 17D Y E L L O W FE\rER VIRUS T O MOUSE BRAIN BY SERIAI, PASSAGI" BV
('aptain 1'. 1). I\It'2ERS, Royal Army ~,lcdic;fi Corps *
i From the Federal Laboratory ?~'ervice, Headqztart~* ~, ~,'aba, Logos, X&eri,:~)
T h e p r o d u c t i o n at Yaba, Nigeria, of a 17D yellow fever m o u s e - b r a i n vaccine, for use in h u m a n i m m u n i z a t i o n by scarification, p r o m p t e d an e x p e r i m e n t designed to investigate the b e h a v i o u r of the 17D virus on serial passage in m o u s e brain. It was t h o u g h t that this m i g h t lead to a strain of yellm~ fever virus which, while still c o n f o r m i n g to the W H O req u i r e m e n t s for 17D c h i c k - e m b r y o seed vaccine (and being, therefore, in all p r o b a b i l i t y safe for use as an i m m u n i z i n g agent), would yield a higher titre in m o u s e brain, giving a cheaper vaccine. At the same time it was felt that such a strain m i g h t also p r o d u c e a significantly greater c o n v e r s i o n rate a m o n g s t those vaccinated t h a n that p r o d u c e d bv 17D chicke m b r y o or the first passage 17D m o u s e - b r a i n vaccine in use at present. T h i s work is r e p o r t e d in two parts, the first dealing with the changes i n d u c e d in the 17D virus as d e m o n s t r a t e d in mice, the second dealing with the changes d e m o n s t r a t e d in I n d i a n rhesus m o n k e y s ( 3 t a c a c a mulatta).
1. TIlE CHANGES IN THF VIRUS OBSERVED IN MICE MATERIAI.S AND METHODS Notation The term " 17D-MA " (the letters MA standing for mouse adapted) refers to the 17D vellmv fever virus grown in mouse brain. As a suffix to this is added the passage lexel, rims 17D-'AL~-I refers to virus after one mouse-brain passage, that is, material harvested from mice suffering a specific 17I) yellow fever encephalitis as a result of the intracerebral inoculation of 17D chick-embryo grown virus. 17D-.\IA-2 is material harvested from mice whep paralyscd following the inoculation of 17D-MA-t material, and so on.
Bur/Fred normal saline ( BI :\',5') This was prepared by adding sulticient sodmm chloride to produce a concentration of 0 85 per cent. in Sorensen's M15 phosphate buffer solution, pH 7.4. It was sterilized in the autoclave. 0.75 per cent. Bovine albnmin saline (B,4/S) A stock solution was prepared by dissolving Armour brand bovine albumin (fraction V) in B/NS to make a 7.5 per cent. solution, which was filtered throut~h a seitz pad. For use, this was diluted to 0.75 per cent. with further B / N S . * Thanks are due to Sir Samuel Manuwa, Chief Medical Adviser to the Federal Government of Nigeria, for permission to publish this paper. I am grateful to Dr. F. N. Macnamara, Director, West African Council for Medical Research, who has given a great deal of advice. Mr. R. C. Kent. Messrs. l,. D. Jaja, and H. Imemogha rave techMcal assistance.
44(~
\ I ) A I ' T A T I O N OF 171) YELLOW FEVER VIRUS "10 MOUSE BRAIN
Mice Mice were derived from the nlousc colony attached to the Yellow Fever Vaccine Laboratory. T h e colony is of Swiss albino mice, obtained from the Virus Research Institute, Entebbe, Uganda. T h e standard " group " of mice numbers six individuals, taken at random from those of the age-group concerned.
The viruses T h e 17D yellow fever virus employed was derived from vaccine lot 1982 nlade m the Rockefeller Laboratories, New York. From this was prepared in chick embryos a primary seed lot (PSi) which has been in use for the preparation of secondary seed lots for vaccine production in this laboratory, and which was the starting point for two of the passage series reported. T h e third passage series was initiated with secondary seed lot S2/I)S.,, prepared in chick embryos inoculated with material from primary seed lot PS,,. T h e Dakar vaccine strain of yellow fever virus was derived from I)akar vaccine batch A98, alnpoules of which were obtained from the West African Council for Medical Research. This was used as it was, or, as the titre was low due to prolonged storage, after one further passage in mice.
Passage details Of the three passage series, the two initiated with the PS 2 virus were designated PS., 1 and PS,,/2 respectively. T h e third series, with the S2/PS., starting point, was called the S2/PS 2 series. Twelve groups of mice were used at each passage level in the I)S,j1 and PSi/2 series, and six at each level of the S2/t S~ series. T o initiate the passages, the contents of an ampoule of the appropriate virus was rehydrated with B/NS., and inoculated in 0.03 ml. amounts intracerebrally into mice 35 days old. T h e first mice to become paralysed as a result of yellow fever encephalitis were separated, chloroformed, and the brains removed aseptically. T h e brains (numbering between three and six--these figures being adhered to throughout the passages) were placed in a sterile mortar with a little powdered neutral glass, and ground, adding slowly 3 ml. of BA/S per brain. When a fine emulsion had been produced, it was poured into a sterile universal container, and stood in a refrigerator (plus 4°C.) for 1 hour. One ml, of the supernate was then removed, and diluted to 1:10 by the addition of 9 ml. BA/S. This approximate 1:100 dilution of mouse brain material was used as the inoculum for the next group of mice in the series, and, where applicable, as the 1 0 for titration. This procedure was used unchanged for all the passages in all three series, except that in the PS._,/2 series some of the supcrnate before dilution to 1:100 was ampouled in {I. 5 ml. amounts, and freeze-dried, for subsequent reference. Titration of the 1:100 passage material was carried ont at each level in both the PS2/1 and PS2/2 series. In this fashion, the PS.j1 and $2,'1 S 2 series were continued for 28 consecutive passages each, and the PS,,/2 series for five passages. Care was taken to maintain accurate records of the individual mice, the mouse record cards being marked at the same time each day. T h e mice were observed for 21 days, those falling paralysed first being the ones used for passage purposes. In calculating the axerage survival time (A.S.T.) these mice were considered to have died on the day on which they were used for passage. Any mouse falling sick more than 2 days before the retnainder in tile group, was discounted for A.S.T. calculation. T h e amount of virus contained in the 1:100 mouse brain passage material was such that 100 per cent. of mice inoculated with it, (tied. T h e age of the mice used was nearly ahvays 35 days on the (lay of inoculation. If
P. I). MFV.RS
447
insufficient 35-day-old mice were available on a given day, tile deficiency was made up with mice 34 or 36 days old. The limits of age used were 32 to 38 days. Not less than {t() per cent. of the mice were, however, 35 days old exactly when inoculated.
Titrations Tenfold dilutions of the material to be titrated were prepared in BA/S. These were inoculated in 0.03 ml. amounts into not less than one group of 35-day-old mice per dilution. The 50 per cent. end point was calculated accordin~ to the method of REED and MUEN('II (1938).
The reference levels As it was obviously impracticable t{) investigate exhaustively the character of each of the viruses obtained at each passage level, certain " reference levels " had to be chosen. These were the 17D-MA-1, 17D-MA-3 and 17D-MA-17 viruses. Chick-embryo grown 17D virus and the strain of French neurotropic yellow fever virus used in the preparation of the Dakar scarification vaccine were added for comparison, as " controls."
The preparation of the reference level materials 17D-MA-1. :\ series of batches of 17I) mouse-brain vaccine produced routinely by the laboratory for human immunization by scarification were used. This vaccine is identical with 17D-MA-1 material, being produced by a development of the second method described by CANNON and DEWI:IURST (1955), from the brains of mice inoculated with 17D chickembryo seed virus.
17D
448
ADAPTATION
OF
171) YELI,OVv' FEVER VIRUS TO
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Fig. 1, The changes observed in the Average Survival Time of adult mice m three serial passage experimems with the 171) yellow fever virus.
Average survival time calcu&tion T h e A.S.T. of 35-day-old mice following intracerebral inoculation with the various viruses under consideration was investigated in two distinct ways. Firstly, as the passages proceeded, the average time between inoculation and death was calculated at each passage level for each of the three passage experiments. Secondly, to allow for the effect of virus dose on A.S.T., each of the reference level viruses, and the two " control " viruses, were subjected to a series of t:~trations (10 for the Dakar vaccine strain, 20 each for 17D-MA-3 and 17, and 30---not done specially, but extracted from vaccine production records- for 17D-MA-1 and 17I) chick-embryo vaccines), l ' r o m each titration, the A.S.T. at each dilution was calculated, and the results from each of the separate materials grouped according to the log of the \irus dose, to the nearest whole n u m b e r (Table). In this connexion, only mice dying of a characteristic yellow fever encephalitis were included in the calculations. This is contrary to usual practice, which has been to allot some arbitrary figure for survival time to those mice remaining alive at the end of the experimental period, and including this ']'ABLE. The average survival time of 35-day-old mice inoculated intracerchrally with five different strair, s of yellow fever virus, related to virus dose. Average surx ival time in days, with : Log virus dos(,
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T h e Average Survival T i m e of adult mice inoculated with various strains of yellow fever virus, related t'.) virus dose.
figure in A.S.T. calculations. Justification for the change is found in the clear-cut picture obtained in Figure 2. It will be seen that the final average of the 10, 20 or 30 observations at each of the logarithmic steps of virus dosage is obtained (at all but the highest dilutions) f:rom n o t less than 60 individual mice (for the I)akar strain) or 120 or 180 for tl~e r~thers.
Reactions of immature mice As an extension of the above, it was decided to discover tile mortality ratios of immature mice inoculated extraneurally with varying dosages of the various rd'erence level viruses and controls. For the purposes of the experiment, two age-groups were selected " younlz mice " (13-15 days old), and " infant mice " (3-5 days old). The procedure ;~as to prepare serial tenfold dilutions of the material under test in I{A S., each dilution being inoculated intracerebrally into one group of adult (35-day) mice for titration of tile agent. Next, the dilutions x~hich it was desired to test were inoctflated into immature mice (0.f16 ml. intraperitoneaily into young mice, and (I./13 ml. subctEtancouslv into infant mice), 10 groups of young mice, and four groups of infant mice c,sually being e,,nployed at each dilution, though the exact number varied with thc availability of mice of ti~,: correct age. The mice were observed daily for 21 days, individual records being kept. This procedure was repeated as required with all the virus materials, with the excepti(,~ (~f t71)-5IA-3, which was not lcsted in i m m a t u r e m i c e . RESUI:]'S
lver~w survival times Figure I illustrates graphically the changes in A.S.T. in each of the three passage experiments. The first two points in the S,,,:PS,, series arc omitted, as, due to insufficient mice, only three groups were used for each of the first two passages of this series. Included il~ the lower part of l:igurc 1 are the values for the titrcs of the passage materials observed in the PS.~/1 and PS2/2 series. The table gives the results of the A.S.T. calculations for the reference level and " control " viruses, related to virus dose as described above. Fi~zure
450
ADAPTATION
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2 expresses these graphically by plotting the 1o~ of the virus dose against the reciprocal of the A.S.T. in days. Mortality ratios in inzmatu;'e mice Figures 3 and 4 show graphically the mortality ratios in young and infant mice (as defined above), respectively, subjected to varying dosages of the reference and control viruses. These results are not given in greater detail, as, contrary to expectations, they proved to be of little value in the present context. MORTALITY
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P. I). MEI.:RN I|.
431
THE CHAN{;ES IN TIlE VIRUS OBSERVE[) IN RHESUS MONKEYS (]Flacaca mu/atta) MATERIALS AND METHODS
Materials and methods dcscribed in section 1 of this paper should be referred to fm~ details not included here.
Monkeys All the monkeys used were rhesus monkeys (31. mulatta) received from India in the same consignment, 6 months before the experiments recorded here were begun. At the time they were used, they were apparently fit, and their average weight x\as 4!~ lb. (2. {) kg.), with limits between 3~ and 5½ lb. (1.7 to 2.5 kg.).
Inoculation and subsequent handlin;: The animals were separated into individual cages 7 days beforc their inoculation. From this time on, they were examined, weighed, and had their rectal temperatures recorded daily. On the day of inoculation, the monkeys were bled from the femoral vein to obtain serum for a pre-inoculation mouse-protection test, and the scalp was shaved. They were then anaesthetized with nembutal administered intravenously, and the skin and skull over the left frontal lobe perforated after the application of spirit by means of a dental burr held in the handpiece of a dental machine. The inoculum (which was reconstituted a;ld diluted in B/NS. to give an approximate dose per 0.25 ml. of 10,000 adult mouse IA)a ), was kept on ice, and was introduced by means of a tuberculin syringe, 0.25 mh being irmculated per monkey, at a depth of one inch (2.5 cm.). At the completion of the inoculations, the inoculum was titrated in mice 35 days old, two groups of mice beinf_, employed per tenfold dilution. The monkeys were bled on the 2nd, 4th and 6th days following inoculation for demonstration of circulating virus. The blood was allowed to clot, and was lightly centrifuged to obtain serum. This was inoculated intracerebrally undihtted, and diluted 1:10 and 1:100 in B/NS., into not less than one group of 35-day-old mice per dilution, 0./)3 ml. being inoculated per mouse. The mice were observed for 21 days, deaths from characteristic yellow fever encephalitis being recorded. Scrum virus titres were calculated by thc mcthod of REED and MUENCH (1938). The experimental period terminated on the 30th day after inoculation, when the surviving monkeys were bled to obtain serum for a post-inoculation protection test, and returned to the main pens. Those monkeys which, on their daily inspections, showed any change in bcha~iotlr, or appeared weak, were singled out for special attention. Daily records of their clinical condition were maintained, and, if and when they became so sick as to appear moribund, they were killed. The brain, and in some instances, the spinal cord, were removed. Duplicate contiguous pieces of the cerebrum, thalamic nuclei, cerebellum, pons, and, sometimes, cervical and lumbar cord (the samples from the brain being taken from the side remote from the site of inoculation) were collected. One of each pair was placed in fixative for histological examination, the other in a sterile container, and stored at minus 65~C., for virus titration. After removing the brain, each monkev was submitted to full postmortem examination. At a convenient time, the pieces of brain for virus titration were thawed, and emulsified by grinding with powdered glass, adding B,:NS. to make a 1:2 dilution of brain material. The
452
AI)APTATION OF ] 7 i ) YELLO\~" I,'I,]VER VIllUS 1'(7 MOUSE B R \ I \
resultant suspension was stood in the refrigerator for 10 minutes, the supernate being designated 10 °, titrations being carried o u t in tenfold dihltions, onc group of 35-day-old )nice being employed at each dilution. Twenty-one monkeys were available at the commencement of the expcrhnental period. Of these, six were used to test the 17D-MA-17 virus, six for the 17D-MA-1, six for 17I) chick-embryo vaccine, and the remaining three for the 17I)-MA-3 virus. Of these, one of the six inoculated with 171)-MA-I material died immediately following inoculation.
Protection tests T h e pre- and post-inoculation serum samples from each of the monkeys which survived the experimental period were paired, and with the pre-inoculation sample from those that were killed, were subjected to protection testing. T h e test used was the SMvrHm:R:'< (1945) intraperitoneal " 1 per cent. virus " test in immature mice. Each serum sample was put up undiluted, and at a dilution of 1:4 in I}A/S.
RESULTS
Virus dose Titration of the inocula employed gave the following rcsuhs, expressed in i.1)-.,)inoculated per monkey : iA)su 17D chick-embryo material 12,08(i 17D-MA-I 8,53~) 17D-MA-3 16,6()() 17D-MA-17 5,90() (~h'eulating ,:irus All the monkeys showed circulating virus ,m one (;r more of the three occasions on which each was tested. T h e level of circulating virus was nearly ahvays 1.w the commonest positive finding bcing a " trace," that is, less than one LD~) per 0.03 ml. of undiluted monkey serum. T h e highest titre fo,,md was one of 60 Ll)a0 per 0.03 ml. of serum. No pattern could be deduced from the rcsults, and there appeared to be no relationship betx~een the amount of circulating virus demonstrated, and 1he strain or titre of the inocuh]m.
Accepting as the upper limit of normal for tile rectal temperature of rhesu:; monkeys a figure of 104 <~F. (4().0"C.), all the monkeys inoculated showed a febrile response. T h e fever lasted for between 3 and 6 days, the lowest maximum temperature recorded durh~g the febrile period being 105.0°F. (40.5%:.), the highest 106.6°I r. (41.4°C.). No relationship u as observed between the duration or height of fever and the virus inoculated, though those animals x~.hich suffered a severe cncephalitis generally had a brief period of fever, followed. at or just after the onset of encephalitic symptoms, by a dramatic fall in temperature of between 2°-7:~I". (1.1-3.9°C.) over a period of 1 or 2 days. T h e more severe the signs, the lower the temperature ; those monkeys with a mild encephalitis ha\ing a smaller drop, the temperature rising agaiu to between 103 and 1()4 F. (39.4-4().():C.) as recovery commenced. The only wav in which the fever occurring in the monkeys could be linked with the virus
p. t>..xlEH~s
453
causing it was that there was a variation in the average time of onset of fever in relation t() the virus inoculated, thus " Average time between inoculation and onset ,)t t'e',cr, in days I) ,'q
171) chicl{-cml~r?,,)material 17D-MA- 1
~) .4 5 .() 4.
l 7D-MA-3 171)-MA-17 T h e s e fi~ures are expressed ~zraphically as a part ()t" Fi~.~urc 5.
~ct/BArton
I ~ I D [ N C E OF E~EPH~LITIS,
P[RIOD
°/o
~O 1 aO
I XE'Y , N C I D E N £ [ OF [ N C E P H A L I T I 5 - -
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! NC UBAT)3N P [ RIO0
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MOUSE ~,I~A)N
PASSA~
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Fig. 5. T h e i1~.:idencc of encephalitis and the i n c u b a t i o n peri,)d in rhesus m o n k e y s inoculated ~ i t h various 17D derived strains of vellmv fever virus.
Encephalitis T h e onset of encephalitis was in exerv case but one marked by increasing drowsiness (in the lone case it was marked by tremor), which was followed in some 24 hours by increasing weakness, occasionally accompanied by minor convulsions. T h e weakness usuallv started on one side of the body, spreading, in the more severe cases, to the other. .\s the weaknc:;s increased, the animal lapsed more frequently into semi-coma, the final state being a deeply comatose, flaccid animal, which was, at this stage, killed. In those monkeys which recovered there was always a sufficient degree of hemi- or quadri-paresis accompanied by severe drowsiness or semi-coma to enable ~l confident diagnosis to be made. T h e period between tl~e onset of the s y m p t o m s and the arrival at an apparently moribund state varied between 3 and 5 days. In those animals which wcre not killed, recovery took place in between 5 and 2() days, though one was left with a residual right sided hemiparesis. As there was such a wide x ariation in the incidence of encephalitis between animals inoculated with the various strains under considcration, they will be dealt with separately. T h e incidence is expressed graphically as part of Figure 5.
454
ADkPTATION
OF
17D
YELI,OV~ FEVER VIRUS TO MOUSE BRAIN
17D chick-embryo material
No cases of encephalitis were observed in the six monkeys inoculated with this material. To this result may be added the results of routine safety tests on batches of 17I) chickembryo seed virus. The records of two such tests (all that could be located in detail) revealed that 12 monkeys had been employed, all without evidence of encephalitis. As all these tests were on the seed virus PS., or on secondary seed lots prepared from it, it is apparent that the strain of chick-embryo grown 17D yellow fever virus in use at Yaba is, to the extent to which it has been tested, non-encephalitogenic no cases of encephalitis having been recorded in 18 monkevs which have been employed in safety testing it.
l 7D-MA- 1 One of the six monkeys inoculated with this material, died immediately after inoculatiom Of the remaining five, one developed mild encephalitic symptoms on the 8th post-inoculation day, returning to apparent normality by the 13th. The results obtained from the two monkeys inoculated with the 17D-MA-1 virus referred to by CAXXO,N"and DEWHURST (1955), (during the original safety testing of 17D mouse-brain vaccine), neither of which developed encephalitis, may be included here. Thus the 17D-MA-1 material produced one case of encephalitis in seven monkeys inoculated, an incidence of 14 per cent. "7 "~ 1/D-MA-.~
Only three monkeys were inoculated with this material. Of these, tx\o developed encephalitis, both on the 7th post-inoculation day. One of these was killed on the l lth post-inoculation day to confirm the aetiology of the condition, although it is probable that the animal would have survived. The other had returned to normal, thongh with a residual right-sided weakness, by the 26th post-inoculation day. The incidence of encephalitis with the 17D-MA-3 virus was, therefore, 67 per ccnt.
17D-MA-17 Of the six monkeys inoculated with this material, all (100 per cent.) developed encephalitis between the 6th and 9th days following inoculation. Onlv one was not killed : it had returned to normal 11 davs later. Histological exami~tation I am indebted to Dr. W. G. C. Bearcroft, West African Council for Medical Research, who very kindly carried out the histological examinations. Specimens taken from all the monkeys showed lesions typical of a viral enccphalo-myelitis. The lesions were present predominantly in the cord and hrain stem, to a lesser extent in the cerebrum : thev were absent from the cerebellum. Uirus iso&tion Virus was isolated from the brains of all six monkeys killed. In nearly every case, most virus was found in the tbalamic area. The impression was gained that the amount (ff virus fell rapidly as death approached the highest titres (up to 23,(t00 LDa~) per (}.03 ml. of brain material prepared as described) being found in the thalami of animals killed in the earlier stages of their encephalitis. There was no good correlation between the severity
t'. 1). MEERS
4.,5
of damage seen histologically, and the virus titre. The cerebellum, in most cases, was found to contain fair amounts of virus, although no histological lesions could be demonstrated. Protection tests
All frst (pre-inoculation) samples of serum were found to gi~e a negative (non-protective) protection test result. All the samples collected on the 30th day gave a positive (protectiw:) result undiluted, and all but one (which gave an inconclusive result) were positive at a dilution of 1:4.
I)ISC[-SSION
The safety of a 17D velh)w fever vaccine produced ira mouse brain has been questioned on three counts. These are, firstly, the introduction by vaccination, in addition to the 17D virus, of pathogenic organisms, particularly viruses, which might have been present unsuspected in the brains of the mice from which the xaccine was prepared. Secondly, the production of a~l allergic demyelinating encephalitis due to the iutroduction of foreign neural tissue. Thirdly, and most important, the risk of a dangerous increase in neurotropism of the immunizing virus, with the attendant danger of causing encephalitis. 'Fhesc criticisms have been discussed very briefly before (MEt.:I~s, 1957) it is with a more detailed examination of one of them, that dealing with increased neurotropism, that we are concerned here. Two criteria generally applicable to the adaptation of a virus to a new host are, a rising titre of the infective agent, and a shortening of the incubation period, with succeeding passages, until adaptation is complete. The adaptation of the 17I) yellow fever virus to mouse brain is no exception to this, a marked early rise in titre of the passage lnaterial being seen in Figure 1. That there is a true reduction in ineubatio)l period (measured in this instance by the aw~rage survival time) cannot be deduced from Figure 1, as the rising titre of itself would produce a shortening of the A . S . T . , but is clearly dclnonstrated when the A.S.T. is related to the titre of the im)culum, as in I-'igurc 2. "1'he rapidity with which both the criteria of adaptation change in the first five or six passages is most striking, and ser\es t() underline once again the considerable lability of the 17D virt,s. That these observatio)~s are not the result of a freak mutation is x;cll shown in 1,'igure 1, where the similarity of response in the three passage experiments can be seen. It would appear from this that the course of events in the adaptation of the 17D virus t() mouse brain is constant, and reproduciblc at will. The hoped-for clear-cut " spectrum " ()f mortality ratios in immature )nick, increasing the further the passages got away from the 17D chick-embryo virus, and firmllv arriving at the high mortality expected with the i)akar vaccine strain, did n()t occur, 'l'}~e Dakar virus lived up to expectations, killi,ag 100 per cent. of infant mice at very loxx dosage levels (Fig. 4), and producing (l:ig. 3) a sp.,,ooth, steadily rising curve of mortalities with increasing virus dosage, in young mice. F(~r all the other viruses examined in young mice, however, the results obtained showed widely differing mortality ratios with similar dosages of the same agent on different occasions and, apart from noting that all the strains tested were, in generai, less pathogenic than the Dakar strain, no other deduction is possible. With infant mice, there was a fairly well marked difference between 17D chick-embryo and 17D mouse-brain vaccines on the one hand, and 17D-MA-17 on the other, in that the latter approached closer to the observed mortality ratio f(,r the Dakar strain. In passing it is of interest to note that
456
ADAPTATION Ol,' I 71) YELLOD, FEVER VIii.US '['O MOUSE BRAIN
the unmodified 17D virus is capable of producing mortality in immature mice on extraneural inoculation, contrary to the generally held opinion. Before the safety tests in rhesus monkeys were started, the production of a \ accine from the mouse adapted virus at a passage level higher than the first (that is, the cxisting 17I) mouse-brain vaccine in use at present) appeared attractive. Not only had the titre risen, making the hoped-for cheaper xaccinc a possibility, but also the virus had become more pathogenic to mice, giving rise to the hope that a greater conversion rate among those vaccinated might result. The metlnod by which the safety of a given yellow few'r immunizing agent may be investigated is well known, and is routine practice in several laboratories throughout the world. The assessment of the results obtained, ho~xever, rests entirely on the very few reports of accidents involving the appearance of recognized cases of encephalitis following vaccination in man, and relating this to the bchaviour of the causal virus in safety tests in rhesus monkeys. The safety of a given strain of yellow fever virus intended for human immunization is, therefore, judged on standards which are somewhat arbitrary. In the present instance, as the criterion of safety, the U.N.R.R.A. regulations (as subsequently adopted by WHO) for 171) chick-embryo seed vaccine testing in rhesus monkeys were accepted. These (WHO, 1956) lay down limits for circulating virus (as an index of viscerotropism), production of immunity, ai~d incidence of encephalitis (as an index of neurotropism), in rhesus monkevs. Not less than six lnonkevs arc required to be used for each test. More recently, a new set of draft regulations has been published (WIIO, 1957), which proposes the increase of the minimum nulnber of mo.'nkcvs to be used, to 10 per test. In either case, the requirements as to circulating virus and the production of immunity are met by all the strains of virus tested here, and it is only the neurotropism of the various strains which is of interest. The incidence of encephalitis permitted by the regulations still in force is 33 per cent.that is, in two monkeys out of six. 'l'he draft regulations reduce this figure to 20 per cent.two out of 10. The 17D-MA-1 virus, with an observed incidence of encephalitis of 14 per cent. (one out of seven) is well inside both these limits. Thus, although the total number of monkeys tested is still small, the 171) mouse-brain vaccine fulfills all the criteria of safety for a 17D chick-embryo seed virus. All the other viruses tested, however, exceed very markedly the upper limit of what is considered safe, and thus any hope of using the mouse adapted 17D virus for human imnmnization bey(rod its first passage in mouse brain is dashed. The increasing incidence of encephalitis in rhesus monkeys as adaptation to mice of the 17I) yellow fever xirus proceeds, reflects closely the changes observed in the average survival time in mice recorded in the first part of this paper. Thus the early rapid change noted in mice is accurately mirrored in monkeys (Fig. 5), both as regards the incidence of encephalitis, and the length of the incubation period to the onset of fever. It would be interesting to know if the various 17D seed viruses in use throughout the world for the production of yellow fever vaccine (which are known to vary in encephalitogenic properties as demonstrated in rhesus monkeys) could, if tested in one centre, be placed in order on a scale of increasing neurotropism, and if mouse A.S.T.'s could be similarly graded, as has been done here, producing a picture like that seen in Figure 2, on a smallcr scale. If this were done, the " best " 17D substrain could then be chosen for use by all vaccine producing laboratories. It is a pity that the number of monkeys available for this work was so limited. The incidence of encephalitis with the Dakar vaccine strain of virus could not be demonstrated, and, although work on this aspect of the French neurotropic virus has been done (LLOYD
I'. i), MEt:,It>
457
and Iq.;NNa, 1933) there is, potentially at least, a difference between the virus tested by them and the strain of virus used at present for vaccine production at Dakar. It would also have been preferable to have used ,'note moJ~kevs to test each of the existing " refcrcnce level " viruses, particularly i 7 D - M A - 3 , :rod to have inserted more such reference strains, say at 17D-MA-2, and at the 5th and t0th passage levels, i-lox\ever, the fact that the results fit so xvcll into a smooth pattern (l:ig. 5), which agree closely with the rcstdts ~btained in mice, suggests very str,.mgly that the recorded results are not too far from those which would have been observed had more experimental animals bccn available. Although the work reported failed in its primary objcctive of providing a 1~c~ substrain of the 17I) virus, safe (according to W I t ( ) requirements) for h u m a n immunization, it has thrmvn considerably ~>orc light OI'i the safety ) f first passage 17D mo,dsc-braira vaccine as produced at prcscr~t for scratch vaccinatitm, it has als~,~ demonstrated ,~/c<: avain the considerahle lability of the " fixed " 17D strai,~ {,1: xcllow fever virus. ~I:MMARY T h e course of events in the adaptation t~f the 171) vclhm fever ~irus to mouse brain is described. T h e changes in its character as demonstrated in Swiss albino mice, and in rhesus monkeys, are recorded and compared with the bchaviour of 17D chick-embryo grown virus, and the I)akar vaccine strain of the l:rench neurotropic virus. T h e experiments showed that the neurotropic character of the virus underwent a marked, rapid change. After 17 passages in mouse brain its neurotropism had increased sufficiently to cause an incidence ~Jf encephalitis of l(}l) per cent. in rhesus monkeys inoculated with it. P,EI:ERENCES CANNON, D. A. & I)EWHUt