Archives of Virology

Archives of Virology 61, 273--281 (1979)

© by Springer-Vertag 1979

Selective Inactivation of Hemagglutinin and Neuraminidase on Mumps Virus By ]~OLA~CD~ L~PRAT and M I C ~ z E AYMARD Laboratoire de Baetdriologie-Virologie, Universitd Claude Bernard, Lyon, France With 2 Figures Accepted June 11, 1979

Summary The thermal stability and the effect of guanidine on the hemagglutinin and neuraminidase of three strains of mumps virus were compared. The heat inactivation of hemagglutinin resulted in the concomitant, loss of nenraminidase. The effect of guanidine at various molarities showed t h a t the neuraminidase was more sensitive than the hemagglutinin and a selective inactivation was obtained after exposure to 1.5 ~r guanidine. However differences in sensitivity of both activities (hemagglutinin and neuraminidase) to heat and guanidine inaetivations were observed among strains and correlated with differential susceptibility to non-specific inhibitors of the strains.

Introduction The mumps virus is a budding RNA virus with glycoprotein projections sticking out of the viral envelope. On mumps virus (11) and other paramyxoviruses SV5 (17), Sendal (20), NDV (18) both activities hcmagglutinin and neuramin~dase reside on the same glycoprotein. Compared with influenza viruses and other members of the paramxyovirus group, the hemagglutinin and the neuraminidase of mumps virus were shown antigenically specific of the virus (3) and until now, no antigenic variation has been demonstrated among m u m p s virus strains neither b y neutralization nor by H I tests. Different NDV strains, which do not show major serological differences, have been discriminated b y several biochemical parameters: thermal inactivation rates of hemagglutinating and neuraminidase activities (16) and analysis of structural proteins by PAGE, isoelectric focusing and fingerprint of tryptic peptides (8). For mumps, previous studies (4, 19) have reported differences between strains of mumps virus based on their different ability to agglutinate various species

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e r y t h r o c y t e s . M o r e r e c e n t l y , d i f f e r e n c e s i n t h e r m a l s t a b i l i t y of i n f e c t i v i t y (50 ° C/ 30 m i n u t e s ) h a v e b e e n repozCed (9). Y e t , l i t t l e is k n o w n a b o u t v a r i a b i l i t y of t h e strains in their sensitivity to the non-specific inhibitors. I n t h i s p a p e r , s o m e of t h e p r o p e r t i e s of t h e N a n d t t of t h r e e s t r a i n s of m u m p s virus are compared: thermal inactivation rate, inactivation by guanidine and s e n s i t i v i t y t o t h e n o n - s p e c i f i c i n h i b i t o r s of t h r e e a n i m a l species.

Materials and Methods Virus T h r e e s t r a i n s of m u m p s v i r u s i s o l a t e d f r o m 1952 to 1973 d e s i g n a t e d BOS, C A R a n d F E R were utilized. T h e B O S s t r a i n was i s o l a t e d in 1952 f r o m t h e s a l i v a of a child w i t h p a r o t i t i s a n d w a s p r o p a g a t e d for m o r e t h a n 50 p a s s a g e s i n e m b r y o n a t e d c h i c k e n eggs. T h i s s t r a i n w a s a d a p t e d t o a l l a n t o i e c a v i t y a t t h e 14th passage. T h e C A R s t r a i n was i s o l a t e d i n t 9 6 8 f r o m a p h a r y n g e a l s w a b t a k e n f r o m a ehitd w i t h m e n i n g i t i s a n d was u s e d a t t h e 41st passage i n a m n i o t i c c a v i t y of e m b r y o n a t e d c h i c k e n eggs. T h e F E I ~ s t r a i n , o b t a i n e d i n 1973 f r o m t h e p h a r y n g e a l s w a b of a child w i t h m e n i n g i t i s , was p r o p a g a t e d i n e m b r y o n a t e d c h i c k e n eggs in t h e a m n i o t i e c a v i t y a n d u s e d a t t h e 6 t h passage. T h e t w o l a s t s t r a i n s were n o t a d a p t e d to a l l a n t o i e c a v i t y . F o r t h i s s t u d y , all t h r e e s t r a i n s were g r o w n a t 34 ° C i n t h e a m n i o t i e c a v i t y of 8-days-old fertile c h i c k e n eggs. A m n i o t i c fluids were h a r v e s t e d 5 d a y s a f t e r i n o c u l a t i o n . F l u i d s were clarified b y low s p e e d e e n t r i f u g a t i o n . T h e v i r u s c o n c e n t r a t e d b y eent~Sfugation a t 100,000 × g for 90 m i n u t e s a t 4 ° C w a s h a r v e s t e d in l/a00 of t h e initial v o l u m e , in 0.01 ~z p h o s p h a t e b u f f e r p H 7.2. T h e c o n c e n t r a t e d v i r u s was u s e d as s u c h or s u b m i t t e d t o t w o cycles of p u r i f i c a t i o n o n l i n e a r sucrose g r a d i e n t s [15 to 40 p e r c e n t (w/v) a t 100,000 × g for 25 m i n u t e s a t 4 ° C]. C o n c e n t r a t e d a n d p u r i f i e d v i r a l p r e p a r a t i o n s were s t o r e d a t - - 7 0 ° C.

Neuraminidase Activity (NA ) and Neuraminidase-Inhibition Test ( N I ) N e u r ~ m i n i d a s e t e s t s were p e r f o r m e d a c c o r d i n g t o AYlVlAtCI)-I-IENlCY et al. (2) i n 0.1 5~ a c e t a t e b u f f e r p H 4.6. F o u r s u b s t r a t e s were t e s t e d : f e t u i n [ p r e p a r e d as d e s c r i b e d b y HAM a n d P u c k : (10)], N - a e e t y l n e u r a m i n l a c t o s e (Sigma, 85 p e r c e n t of ~ 2 - + 3 b o n d s a n d 15 p e r c e n t of 2 - + 6 bonds), m u e i n I f r o m b o v i n e s u b m a x i l l a r y g l a n d s (Sigma, 5 p e r c e n t of sialie acid), m u c i n I I f r o m p o r c i n e s t o m a c h (Sigma, 1 p e r c e n t of siMic acid). F o r a m o r e s e n s i t i v e assay, t h e t e s t was i n c u b a t e d for 16 h o u r s at. 37 ° C. N A w a s e x p r e s s e d as o p t i c a l d e n s i t y u n i t s of a c t i v i t y [tile l a s t v i r u s d i l u t i o n t h a t ga.ve s,n O.D. r e a d i n g a t 549 n m of 0.50 d u e t o release N - a e e t y l n e u r a m i n i e acid ( N A N A ) = t O.12). u n i t == 50 n m o l - N A N A ] or as n m o L N A N A released p e r m i n u t e p e r nag of v i r u s protein. A n t i - n e u r a m i n i d a s e a n t i b o d i e s were t i t r a t e d b y t h e e n z y m e i n h i b i t i o n t e s t u s i n g f e t u i n as s u b s t r a t e a c c o r d i n g to t h e W . H . O . r e c o m m e n d e d t e s t (2). T h e a n t i b o d y t i t r e was e x p r e s s e d as t h e reciprocal of t h e l a s t s e r u m d i l u t i o n able t o i n h i b i t 50 p e r c e n t of t h e e n z y m i c a c t i v i t y .

Hemagglutination ( H A ) and tIemagglutination-Inhibition Test (HI) T e s t s were p e r f o r m e d in m i c r o p l a t e s u s i n g p h o s p h a t e b u f f e r ( 5 × 10 -s ~ 1)O4, 0.15 ~ MAC1, p t I 7) as d i l u e n t . E q u a l v o l u m e s of 0.5 p e r c e n t c h i c k e n or 1 p e r c e n t g u i n e a pig r e d b l o o d cell s u s p e n s i o n were a d d e d to t h e v i r u s d i l u t i o n s a n d set for i h o u r a t r o o m t e m p e r a t u r e . All t i t r a t i o n s were r u n i n d u p l i c a t e . H I a n t i b o d i e s were t i t r a t e d b y a d d i n g 4 H A u n i t s of v i r u s t o d i l u t i o n s of a n t i s e r u m . A f t e r 1 h o u r i n c u b a t i o n

275

Selective I n a c t i v a t i o n of M u m p s Virus N e u r a m i n i d a s e

at r o o m t e m p e r a t u r e , the red blood cell suspension was a d d e d and the test read after a f u r t h e r hour. The H I titre was expressed as t h e reciprocal of t h e highest dilution g i v i n g 50 per cent of inhibition.

Guanidine Treatment Guanidine t r e a t m e n t was assayed as p r e v i o u s l y described (6). Guanidine h y d r o c h l o r i d e (Amino m e t h a n a m i d e HC1, S i g m a grade i) was p r e p a r e d in Tris saline p H 7.3 a n d m i x e d w i t h an e q u a l v o l u m e of virus to give final m o l a r concentrations of 0.5, i, 1.5 a n d 2. T h e virus-guanidine m i x t u r e s were k e p t a t 37 ° C in a w a t e r b a t h for t i m e ranging f r o m 0 to 30 minutes. T h e n the tubes were i m m e d i a t e l y chilled in crushed ice. The t e s t and control (Tris saline in place of guanidine) virus samples were t h e n t i t r a t e d for H and N activities.

Heat Stability and Thermal Inactivations E a c h virus p r e p a r a t i o n was h e a t e d in a swirling w a t e r b a t h at various t e m p e r a t u r e s for v a r y i n g periods of t i m e after equilibration in an ice-water bath. A t a p p r o p r i a t e times, samples were r e t u r n e d to the ice-water b a t h and t h e n assayed for s u r v i v i n g biological activities. The corresponding u n t r e a t e d virus suspension was t i t r a t e d as control.

Non-Specitic Inhibitor Tests The non-specific inhibitors from rabbit, guinea pig sera, allantoic and a m n i o t i e fluids were t i t r a t e d against 4 H A units of virus according to t h e H I m i e r o t e s t and against 2 units of N.

Preparation o/Antisera A n t i s e r a were p r e p a r e d in rabbits a n d guinea pigs. R a b b i t antisera were o b t a i n e d b y inoculating i n t r a m u s e u l a f l y 12,800 H A units of c o n c e n t r a t e d and purified virus w i t h complete F r e u n d a d j u v a n t twice at 4 weeks interval. The animals were bled 2 weeks later. Guinea pig antisera were p r e p a r e d b y simultaneous inoculation of 0.5 ml intranasally and 0.5 ml i n t r a p e r i t o n e a l l y of c o n c e n t r a t e d and purified virus (about 10,000 H A units) twice at 8 m o n t h s interval. The animals were bled 2 weeks later. A reference horse a n t i s e r u m a n t i - m m n p s virus was kindly supplied b y Dr. Chapple, C.D.C. A t l a n t a , U.S.A.

Protein Estimation Virus protein was e s t i m a t e d b y t h e m e t h o d of Lowl~¥ et al. (15) using bovine a l b u m i n as s t a n d a r d .

Results

Relationship o/ Virus Neuraminidase Activity to Hemagglutinin T h e r a t i o of t h e t i t r e s of N t o H was t h e s a m e for t h e t h r e e s t r a i n s ( T a M e 1). Table 1. Relationship o] neuraminidase to hemagglutinin M u m p s strains

H A / 0 . 0 5 ml

NA/0.05 ml

NA/HA

CAR BOS FEI~

102,400 a 51,200 51,200

6,400 b 3,200 3,200

0.06 0.06 0.06

a H A titre expressed as h e m a g g l u t i n a t i n g u n i t s / 0 . 0 5 m l d e t e r m i n e d against chicken I~BC b N A titre expressed as optical density units of a c t i v i t y / 0 . 0 5 m l

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