WORLD HEALTH ORGANIZATION EXPERT COMMITTEE ON YELLOW FEVER VACCINE Geneva 8 - 1 3 A p r i l 1957 P r o v i s i o n a l agenda item 7
ORGANISATION MONDIALE DE LA SANTÉ WHO/YFV/2 28 February 1957 ORIGINAL: ENGLISH
THE MOUSE IBOTECTION TEST FOR YELLOW FEVER by Dr K. C. Smithburn
For any b i o l o g i c a l t e s t which has the widespread usefulness which i s associated w i t h the yellow fever p r o t e c t i o n t e s t , the d e s i r a b i l i t y o f a h i g h l y standardized method Is obvious. The purpose o f the present d i s c u s s i o n i s t o consider whether
s t a n d a r d i z a t i o n o f the t e s t Is f e a s i b l e and what measures i n the d i r e c t i o n o f s t a n d a r d i z a t i o n are p o s s i b l e and reasonable. The yellow fever p r o t e c t i o n t e s t i s u s e f u l i n at l e a s t s i x d i f f e r e n t types of study: 1. Surveys t o study the range and frequency of I n f e c t i o n w i t h yellow fever
v i r u s i n man. 2. Surveys t o discover other mammalian and avian hosts o f yellow fever v i r u s
i n nature. 3. suspect h. F o r the diagnosis o f yellow f e v e r , by t e a t s on paired samples from the case. To determine the time o f appearance and the d u r a t i o n o f Immunity Induced
by v a c c i n a t i o n . 5. 6. strains. I n the f i r s t two o f these categories i t i s imperative that the methods employed be s u f f i c i e n t l y severe t o exclude n o n - s p e c i f i c r e a c t i o n s . I n the l a t t e r four To study the e f f i c a c y o f v a c c i n e s . To study the I n t e r r e l a t i o n s h i p s of v i r u s e s and t o i d e n t i f y newly i s o l a t e d
c a t e g o r i e s the requirement i s f o r methods which are h i g h l y s e n s i t i v e and the
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technical requirements to meet them are not l i k e l y to be s a t i s f i e d by a single method. Since the discovery by Theiler that white mice are susceptible to the neurotropic action of yellow fever v i r u s , no less than eight separate and d i s t i n c t methods have been devised for the conduct of yellow fever protection tests. the intracerebral test In adult mice devised by Theiler himself. The f i r s t of these vao The next vas the
Intraperitoneal proctectlon test i n adult mice previously inoculated Intracerebrally with s t e r i l e starch solution, as proposed by Sawyer and Lloyd. Later, Bugher pointed
to certain short-comings of the Intracerebral test devised liy Theiler and reported the results of his experiments to meet these. The r e s u l t was a considerable E a r l i e r workers had
reflnenent of the intracerebral adult mouse protection t e s t .
found that Infant mice are very much more susceptible to yellow fever virus than a d u l t s . Nevertheless, the techniques employed In the early protection test experiments on yellow fever virus Involved the use of adult mice, whether the Inoculations were given by the Intracerebral w the intraperitoneal method. Then Whitman found that the
s u s c e p t i b i l i t y of Immature mice was s u f f i c i e n t l y great that a satisfactory test could be done by intraperitoneal Injection In mice 21 days of age without the use of the Intracerebral inoculation of starch which Sawyer and Lloyd's method involved. In
East A f r i c a , two different sensitive methods were taken up, one of these employing Infant mice, the other adult mice, but both involving the intraperitoneal inoculation of a more d i l u t e suspension of virus than most techniques i n use prior to that time. The seventh method which has had very extensive use i s that of the Dakar workers. F i n a l l y there i s the technique employing 17D yellow fever vaccine virus as proposed by Gear and his associates i n the Union of Scout h A f r i c a . With exception of the 17D test proposed by Gear, a l l of the aforeasnttoned techniques Involve the use of neuro-adapted virus of a French s t r a i n . The degree of
neuro-adaptatlon of the virus generally used Is such that the hazard of Introducing I t Into a region where yellow fever does not occur would be minimal,
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One step could therefore be taken toward the s t a n d a r d i z a t i o n of y e l l o v fever p r o t e c t i o n t e s t s by the general use of the same s t r a i n of v i r u s ; moreover I t would probably not
be unreasonable t o expect t h a t a l l workers concerned would be w i l l i n g t o adopt v i r u s of a s i m i l a r passage l ^ v e l and t o agree on the use of e i t h e r l y o p h i l i z e d or frooen, p r e v i o u s l y assayed, stocks o f v i r u s f o r a l l such t e s t s . A l l experienced vorkers w i t h yellow fever v i r u s w i l l doubtless agree t h a t the adoption of a p r o t e i n - c o n t a i n i n g d i l u e n t which w i l l p r o t e c t the v i r u s against t h e d e l e t e r i o u s e f f e c t s of e l e c t r o l y t e s i s e s s e n t i a l . bovalbumin are the d i l u e n t s most commonly chosen. D i l u t e d serum o r a s o l u t i o n o f The use o f serum involves preNevertheless, t h i s i s
t e s t i n g t o determine the s u i t a b i l i t y o f the serum f o r use.
not a serious obstacle and i t i s b e l i e v e d t h a t agreement could be reached among the m a j o r i t y o f workers whether one d i l u e n t , say 10 per cent, non-immune r a b b i t serum I n p h y s i o l o g i c a l s a l i n e , cyr another, say f r a c t i o n V Bovalbumin, was t o be the standard. Experience has shown t h a t the t i t r a t i o n of yellow fever v i r u s i n serum derived from d i f f e r e n t species of i n d i v i d u a l s may give q u i t e d i f f e r e n t r e s u l t s . For Instance,
the v i r u s u s u a l l y gives a s i g n i f i c a n t l y higher t i t r e i n the serum o f non-immune human beings or rhesus monkeys than I t does i n non-immune domestic r a b b i t s . Bugher has
emphasized t h i s f a c t i n h i s c a r e f u l studies of the yellow f e v e r p r o t e c t i o n t e s t and has emphasized the n e c e s s i t y f o r e s t a b l i s h i n g a b a s e - l i n e or norm f o r each species t o be t e s t e d . These f a c t s make i t evident that one of the c o n t r o l s which should
be i n c l u d e d I n every yellow fever p r o t e c t i o n t e s t i s a t i t r a t i o n of the v i r u s In nonimmune serum of the same species o f i n d i v i d u a l s which provide the b u l k o f the t e s t s e r a t o be examined. I n some l a b o r a t o r i e s the procedure i s t o do only a t i t r a t i o n This i s , however, Inadequate
of the V i r u s without admixture w i t h non-Immune serum. as may be seen from the foregoing statements.
Furthermore, the need f o r a t i t r a t i o n
o f v i r u s m immune serum i s obvious i n order t o be c e r t a i n t h a t the i n f e c t i v e agent i s uncontaminated yellow fever v i r u s . These c o n t r o l s should be i n every t e s t . It
i s not s u f f i c i e n t t o prepare a l o t of standard v i r u s , assay I t , and then use I t I n repeated t e s t s without I n t e r n a l c o n t r o l t i t r a t i o n s w i t h i n the t e s t s .
W i t h reference t o the I n t e r p r e t a t i o n o f r e s u l t s there Is l i t t l e doubt that
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p&ge h agreement can be reached i n t h i s . Most vorkers i n t h i s f i e l d already have adopted
the same standards of i n t e r p r e t a t i o n of t e s t s . I t i s w e l l known that d i f f e r e n t s t r a i n s of mice vary enormously i n t h e i r s u s c e p t i b i l i t y to yellow fever and other v i r u s e s . I t i s a l s o known that the same laboratories i n different
o r i g i n a l s t r a i n of mice which has been propagated i n two p a r t s of the world may
a l s o vary i n t h e i r s u s c e p t i b i l i t y to yellow fever v i r u s . of the mice used i n jcellow fever p r o t e c t i o Nevertheless, i t should be possible
I t would t h e r e f o r e seem that s t a n d a r d i z a t i o n t e s t s throughout the world i s not f e a s i b l e .
t o reach agreement upon the use of a standard dose of v i r u s which i s e f f e c t i v e f o r whatever mice are used by the l a b o r a t o r i e s concerned. This can be determined In
every t e s t by the t i t r a t i o n of v i r u s which i s done i n the non-immune serum, which w i l l give a c l e a r i n d i c a t i o n of the potency of the preparation i n use In that t e s t and adequacy of the dose present i n the t e s t d i l u t i o n chosen f o r that t e s t . F i n a l l y we come t o the route of I n o c u l a t i o n and the dose, and i t i s upon these In general the
two that the s e n s i t i v i t y or the s e v e r i t y of the t e s t depends most of a l l . the s e n s i t i v i t y of i n t r a p e r i t o n e a l t e s t s i s f a r greater than that of t e s t s and,
intracerebral Inoculation of
i n general, the s e n s i t i v i t y of t e s t s i n v o l v i n g i n t r a p e r i t o n e a l
of Infant mice i s f a r greater than t h a t of i n t r a p e r i t o n e a l t e s t s I n v o l v i n g the use adult mice. Whether the i n t r a p e r i t o n e a l or the i n t r a c e r e b r a l route i s chosen, the
s e n s i t i v i t y or the s e v e r i t y of the t e s t can be v a r i e d by i n c r e a s i n g the dose of v i r u s used i n the t e s t . mitigate against I t i s i n these matters that the a c t u a l o b j e c t i v e s of the t e s t standardization.
In survey work I n v o l v i n g e i t h e r human or animal sera i t i s Important t o make the t e s t s u f f i c i e n t l y severe t h a t n o n - s p e c i f i c p o s i t i v e s w i l l not commonly occur. One
t h e r e f o r e does not r e q u i r e a h i g h l y s e n s i t i v e method and i n the view of t h i s i n v e s t i g a t o r survey purposes are best served by the use of an i n t r a c e r e b r a l technique m adult mice and the adoption of a standard e f f e c t i v e dose of v i r u s of 100 more f o r the species of i n d i v i d u a l from which the t e s t sera are d e r i v e d . U D ^ ^ or It is
b e l i e v e d t h a t the wide-spread adoption of such methods f o r survey purposes would v i r t u a l l y eliminate non-specific p o s i t i v e r e s u l t s and would enable a l l workers t o be
p r e c i s e l y aware of the s i g n i f i c a n c e of r e s u l t s obtained by the workers I n other p a r t s of the world.
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Inasmuch as the m a j o r i t y o f a l l other uses o f the yellow f e v e r p r o t e c t i o n t e s t are f o r purposes In which the d e t e c t i o n of a s m a l l amount of antibody i s Important, i t i s b e l i e v e d that i f s t a n d a r d i z a t i o n i s attempted i t should be on a b a s i s e n t i r e l y d i f f e r e n t frcMn t h a t accepted f o r survey p r o t e c t i o n t e s t s . I n many
of these other uses of the yellow fever p r o t e c t i o n t e s t s . I n t r a p e r i t o n e a l techniques e i t h e r i n a d u l t mice or i n Infant mice w i l l be found much more u s e f u l than an i n t r a c e r e b r a l method i n adult mice. I t would seem best t o l e t the i n d i v i d u a l
i n v e s t i g a t o r s decide upon t h e i r own method of choice f o r these widespread uses o f the p r o t e c t i o n t e s t , and not attempt t o standardize f o r any other than survey purposes.