W O R L D H E A L T H ORGANISATION MONDIALE O R G A N I Z A T I O N DE LA SANTÉ
EXPERT COMMITTEE ON WHO/YFV/2 YELLOW FEVER VACCINE 28 February 1957
Geneva 8 - 1 3 A p r i l 1957 ORIGINAL: ENGLISH P r o v i s i o n a l agenda i t e m 7
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 u s e f u l n e s s which i s a s s o c i a t e d w i t h the y e l l o w f e v e r p r o t e c t i o n t e s t , t h e d e s i r a b i l i t y o f a h i g h l y s t a n d a r d i z e d
method Is obvious. The purpose o f the present d i s c u s s i o n i s t o c o n s i d e r whether s t a n d a r d i z a t i o n o f the t e s t I s 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 y e l l o w f e v e r p r o t e c t i o n t e s t i s u s e f u l i n a t 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 y e l l o w f e v e r
v i r u s i n man.
2. Surveys t o d i s c o v e r other mammalian and a v i a n hosts o f y e l l o w f e v e r v i r u s i n n a t u r e .
3. F o r t h e d i a g n o s i s o f y e l l o w f e v e r , by t e a t s on p a i r e d samples from the suspect case.
h. To determine t h e 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. To study the e f f i c a c y o f v a c c i n e s .
6. To study the I n t e r r e l a t i o n s h i p s o f 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
s t r a i n s .
I n the f i r s t two o f these c a t e g o r i e s i t i s i m p e r a t i v e t h a t 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 f o u r
c a t e g o r i e s t h e 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
WHO/YFV/2
page 2
controls for the t e s t s are often derived from e a r l i e r samples taken from the same i n d i v i d u a l . Thus, the objectives of the t e s t are s u f f i c i e n t l y diverse that the t e c h n i c a l 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 T h e i l e r that white mice are susceptible to the neurotropic a c t i o n 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 f o r the conduct of yellow fever protection t e s t s . The f i r s t of these vao the i n t r a c e r e b r a l t e s t In adult mice devised by Theiler himself. The next vas the Intraperitoneal proctectlon t e s t i n adult mice previously inoculated I n t r a c e r e b r a l l y w i t h s t e r i l e starch s o l u t i o n , as proposed by Sawyer and Lloyd. Later, Bugher pointed to c e r t a i n short-comings of the Intracerebral t e s t devised liy Theiler and reported the r e s u l t s of h i s experiments to meet these. The r e s u l t was a considerable reflnenent of the i n t r a c e r e b r a l adult mouse protection t e s t . E a r l i e r workers had
found that Infant mice are very much more susceptible to yellow fever v i r u s than a d u l t s . Nevertheless, the techniques employed In the e a r l y protection t e s t experiments on
yellow fever v i r u s Involved the use of adult mice, whether the Inoculations were given by the Intracerebral w the i n t r a p e r i t o n e a l 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 s a t i s f a c t o r y t e s t could be done by i n t r a p e r i t o n e a l I n j e c t i o n In mice 21 days of age without the use of the
Intracerebral i n o c u l a t i o n of starch which Sawyer and Lloyd's method involved. In East A f r i c a , two d i f f e r e n t s e n s i t i v e methods were taken up, one of these employing
Infant mice, the other adult mice, but both i n v o l v i n g the i n t r a p e r i t o n e a l i n o c u l a t i o n of a more d i l u t e suspension of v i r u s than most techniques i n use p r i o r 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 v i r u s as proposed by Gear and h i s associates i n the Union of Scout h A f r i c a .
With exception of the 17D t e s t proposed by Gear, a l l of the aforeasnttoned
techniques Involve the use of neuro-adapted v i r u s of a French s t r a i n . The degree of
neuro-adaptatlon of the v i r u s generally used Is such that the hazard of Introducing
I t Into a region where yellow fever does not occur would be minimal,
page 3
One s t e p c o u l d t h e r e f o r e be t a k e n toward t h e s t a n d a r d i z a t i o n o f y e l l o v f e v e r p r o t e c t i o n t e s t s by the g e n e r a l use o f t h e same s t r a i n o f 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 t h e use o f e i t h e r l y o p h i l i z e d or f r o o e n , 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 v o r k e r s w i t h y e l l o w f e v e r v i r u s w i l l doubtless agree t h a t t h e a d o p t i o n o f 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 t h e v i r u s a g a i n s t t h e d e l e t e r i o u s e f f e c t s o f e l e c t r o l y t e s i s e s s e n t i a l . D i l u t e d serum o r a s o l u t i o n o f bovalbumin are the d i l u e n t s most commonly chosen. The use o f serum i n v o l v e s p r e - t e s t i n g t o determine t h e s u i t a b i l i t y o f t h e serum f o r use. N e v e r t h e l e s s , t h i s i s not a s e r i o u s o b s t a c l e and i t i s b e l i e v e d t h a t agreement c o u l d be reached among t h e m a j o r i t y o f workers whether one d i l u e n t , say 10 per c e n t , 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 a n o t h e r , say f r a c t i o n V Bovalbumin, was t o be t h e s t a n d a r d . E x p e r i e n c e has shown t h a t t h e t i t r a t i o n of y e l l o w f e v e r v i r u s i n serum d e r i v e d from d i f f e r e n t s p e c i e s o f i n d i v i d u a l s may g i v e q u i t e d i f f e r e n t r e s u l t s . F o r I n s t a n c e , the v i r u s u s u a l l y g i v e s a s i g n i f i c a n t l y h i g h e r t i t r e i n the serum o f non-immune human beings o r 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 s t u d i e s o f t h e y e l l o w 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 o r norm f o r each s p e c i e s t o be t e s t e d . These f a c t s make i t e v i d e n t t h a t one o f t h e c o n t r o l s w h i c h should be i n c l u d e d I n every y e l l o w f e v e r 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 o f t h e v i r u s I n non-
immune serum of t h e same s p e c i e s o f i n d i v i d u a l s which p r o v i d e t h e b u l k o f t h e 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 o n l y a t i t r a t i o n o f t h e V i r u s w i t h o u t admixture w i t h non-Immune serum. T h i s i s , however, Inadequate as may be seen from t h e f o r e g o i n g 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 t h e i n f e c t i v e agent
i s uncontaminated y e l l o w f e v e r v i r u s . These c o n t r o l s should be i n every t e s t . I t i s not s u f f i c i e n t t o prepare a l o t o f standard v i r u s , assay I t , and t h e n use I t I n r e p e a t e d t e s t s w i t h o u t 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 r e f e r e n c e t o t h e I n t e r p r e t a t i o n o f r e s u l t s t h e r e Is l i t t l e doubt t h a t
WHO/YFV/2
p&ge h
agreement can be reached i n t h i s . Most v o r k e r s i n t h i s f i e l d a l r e a d y 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 t h a t d i f f e r e n t s t r a i n s of mice v a r y enormously i n t h e i r
s u s c e p t i b i l i t y t o y e l l o w f e v e r and o t h e r v i r u s e s . I t i s a l s o known t h a t the same o r i g i n a l s t r a i n of mice which has been propagated i n two l a b o r a t o r i e s i n d i f f e r e n t p a r t s of the w o r l d may a l s o v a r y i n t h e i r s u s c e p t i b i l i t y t o y e l l o w f e v e r v i r u s .
I t would t h e r e f o r e seem t h a t s t a n d a r d i z a t i o n of the mice used i n jcellow f e v e r p r o t e c t i o t e s t s throughout the w o r l d i s not f e a s i b l e . N e v e r t h e l e s s , i t should be p o s 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. T h i s can be determined I n e v e r y 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 g i v e a c l e a r i n d i c a t i o n of the potency of the p r e p a r a t i o n i n use In t h a t t e s t and the adequacy of the dose present i n the t e s t d i l u t i o n chosen f o r t h a t t e s t .
F i n a l l y we come t o the r o u t e of I n o c u l a t i o n and the dose, and i t i s upon these two t h a t 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 . I n g e n e r a 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 g r e a t e r than t h a t of i n t r a c e r e b r a l t e s t s and, i n g e n e r a l , 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 I n o c u l a t i o n of I n f a n t mice i s f a r g r e a t e r t h a n 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 of a d u l t 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 r o u t e 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 . I t i s i n these matters t h a t the a c t u a l o b j e c t i v e s o f the t e s t m i t i g a t e a g a i n s t s t a n d a r d i z a t i o n .
I n survey work I n v o l v i n g e i t h e r human or animal s e r a 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 o c c u r . 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 b e s t served by the use of an i n t r a c e r e b r a l t e c h n i q u e m a d u l t mice and the a d o p t i o n of a standard e f f e c t i v e dose of v i r u s of 100 UD^^ or more f o r the s p e c i e s of i n d i v i d u a l from which the t e s t s e r a are d e r i v e d . I t i s b e l i e v e d t h a t the wide-spread a d o p t i o n of such methods f o r survey purposes would v i r t u a l l y e l i m i n a t e n o n - s p e c i f i c 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 o f the s i g n i f i c a n c e o f r e s u l t s o b t a i n e d by the workers I n o t h e r p a r t s of the w o r l d .
WHO/ïFV/2 page 5
Inasmuch as the m a j o r i t y o f a l l other uses o f the y e l l o w f e v e r p r o t e c t i o n t e s t are f o r purposes I n which t h e d e t e c t i o n of a s m a l l amount o f a n t i b o d y i s Important, i t i s b e l i e v e d t h a t i f s t a n d a r d i z a t i o n i s attempted i t s h o u l d 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 o f these other uses o f t h e y e l l o w f e v e r 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 I n f a n t mice w i l l be found much more u s e f u l t h a n an
i n t r a c e r e b r a l method i n a d u l t mice. I t would seem best t o l e t t h e 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 o f c h o i c e f o r these widespread uses o f t h e p r o t e c t i o n t e s t , and not attempt t o s t a n d a r d i z e f o r any other t h a n survey purposes.
