Archives of Virology 48, 245--252 (1975) © by Springer-Verlag 1975

Antiflenie Comparison of Swine hffluenza Virus Isolates 1 By

Department

D. M. NATH, L. S. I~ODKEY, and H. C. MINOCIIA of Infectious Diseases and Division of Biology, Kansas State University, Manhattan, I~ansas, U.S.A. ~¥ith 4 Figures

Accepted April 14, 1975

Summary Cross hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests demonstrated strong antigenic relationship between A/Swine/Wisconsin/i/73 (8W/73) and A/Swine/Shope/15/31 (SW/31) influenza viruses. An eigthyone fold purification of virus was achieved by adsorption and e h t i o n followed by differential u]tracentrifugation and sedimentation through linear sucrose gradient. Radioimmunoassay using purified :t25Ilabeled viral antigens revealed antigenic variation between the two virus isolates. Neuraminidase of both viruses had p H optima between 6.5 and 7.0, and SW/31 enzyme was relatively more heat stable than SW/73. Introduction

Antigenic drift in influenza viruses involves gradual changes in surface antigens, hemagglutinin (HA) and neuraminidase (NA). The changes may result in mutant viruses possessing altered antigenic sites. In vitro, such mutants are knm~aa to differ in amino acid sequences of polypeptides (8). In major antigenic shift, complete change in one or both surface antigens due to genetic recombination between influenza viruses of different species of animals m a y give rise to new viruses (9, 21). SttOPE (1939), GOMt'ELS (1953), and JEI~-SEN and PETERSON (1957) did not observe antigenic drift in the HA of swine influenza virus isolates (5, 7, t9). However, M~IER-EwEaT et al. (1970) demonstrated a slight but significant antigenie drift in both HA and NA (12). This paper describes a study of the antigenic relationship of swine influenza virus isolates of 1931 and 1973. i Contribution No. 183, Department of Infectious Diseases, and Contribution No. 1239, Division of Biology, Kansas Agricultural Experiment Station, Manhattan, Kansas 66506.

17"

246

D . M . N i : r ~ , L. S. ]~ODKEY,and H. C. M I ~ o c H i :

Materials and Methods Growth o/ Viruses I n f l u e n z a virus strains SW/73 and SW/31 (Hsw l N 1) were p r o p a g a t e d in allantois of 9- to 10-day-old chicken embryos. I n f e c t e d allantoie fluid h a r v e s t e d two days after the inoculation of e m b r y o s was clarified b y centrifuging a t 1500 g for 15 minutes. Virus was pelleted in an ultracentrifuge (50,000g for 3 hours) and resuspended in 0.2 ~ s o d i u m p h o s p h a t e citrate buffer (pH 6.7). Virus was partially purified b y adsorption and elution using chicken red blood cells (RBC).

Preparation o/ Antisera H y p e r i m m u n e sera to viruses were prepared by injecting i n t r a p e r i t o n e a l l y 6- to 8-week-old rabbits with 5000 H A units of virus. E v e r y 10 clays, the animals were g i v e n booster doses i n t r a m u s c u l a r l y w i t h t h e virus m i x e d w i t h F r e u n d ' s ineomplete a d j u v a n t e and t h e y were bled regularly. A n t i s e r u m was i n a c t i v a t e d at 56 ° C for 30 m i n u t e s for t h e neuraminidase inhibition (NI) test and was further t r e a t e d w i t h K a o l i n and chicken R B C for the h e m a g g l u t i n a t i o n inhibition (HI) test (17). Sera from rabbits w i t h a d e q u a t e H I and N I titers were pooled.

H A and H I (i~est H A a n d H I tests were c o n d u c t e d as described b y the W o r l d H e a l t h Organization (22) a n d a d a p t e d to the microtiter m e t h o d b y SEVE~ (18).

Assay o~ Neuraminidase Serial two-fold dilutions of virus were m a d e in saline solut~ion (NaC1 0.85 per cent, p H 7.0). Samples (0.05 ml) of each virus dilution. (in duplicate) were i n c u b a t e d a t 37 ° C for 30 m i n u t e s w i t h 0.05 m i (1.25 rag) fetuin a solution in p h o s p h a t e citrate buffer. N - a c e t y l - n e u r a m i n i c acid (NANA) liberated was assayed according to AMI~-OF~' ( 196 t ). Concentration of n e u r a m i n i d a s e giving 0.5 to 0 . 7 0 . D . at 54:9 m ~ was used as s t a n d a r d dilution in the N I test.

Neuraminidase Inhibition Test Serial two-fold dilutions (0.05 ml) of a n t i s e r u m in saline (2 tubes/serum dilution) were m i x e d w i t h a n equal v o l u m e of s t a n d a r d virus, t h e n i n c u b a t e d at r o o m t e m p e r a t u r e for 4 hours (20). D u p l i c a t e samples of p r e - i m m u n i z a t i o n s e r u m from rabbits were included in the test as controls. The n e u r a m i n i d a s e a c t i v i t y inhibited, d e t e r m i n e d b y the a m o u n t of N A N A liberated, was plotted as percentage of a c t i v i t y of the control sample.

Preparation el Radioactively Labeled Viral Antigens P a r t i a l l y purified virus was s e d i m e n t e d t h r o u g h a 10 to 40 per cent linear sucrose g r a d i e n t a t 35,000 g for 45 minutes. One ml fractions were collected a n d those showing p e a k H A a c t i v i t y were pooled together, dialyzed against p h o s p h a t e buffered saline (PBS), c o n c e n t r a t e d b y u l t r a c e n t r i f u g a t i o n (50,000g for 3 hours), t h e n resuspended in PBS. Purified virus was dissociated w i t h 2 per cent sodium deoxyeholate (DOC) at 37 ° C for 30 m i n u t e s a n d diMyzed against b o r a t e saline buffer (BSB), p H 8.0, t h e n c o n c e n t r a t e d b y e v a p o r a t i o n in dialysis t u b e to a finM c o n c e n t r a t i o n of more t h a n 3 m g protein/ml. P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d using b o v i n e s e r u m a l b u m i n as a standard (lO). The antigen was labeled by mixing it with sodium iodide-lesI in the presence of iodine monoehleride and glycine buffer (Ii). Unincorporated label was removed by dialysis against glycine buffer (0.2 ~, pI-I 8.6). Antigens were clarified by low speed eentrifugation before use. 2 Difeo Laboratories, :Detroit, 2~Iiehigan. s Grand Island Biological Company, Bethesda,

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Antigenic Comparison of Swine Influenza Virus Isolates

247

Radioimmunoassay Various concentrations of unlabeled antigen (0 to 50 y.g) and constant amounts of labeled reference antigen (0.25 ~g in 5 ~l) and antiserum (20 ~1) were mixed (duplicate samples) and incubated for 4 hours at 37 ° C. A predetermined q u a n t i t y of goat antirabbit IgG (0.2 ml) was added; the mixture was further incubated for 1 - - ~ hours at 37 ° C, and then it was kept overnight at 4 ° C. The precipitate was twice washed with cold BSB and dissolved in 0.05 M sodium hydroxide. The supernatant and precipitate were counted for 4 minutes and the percentage binding (12~Iantigen) of the uninhibited control sample was plotted against different concentrations of unlabeled antigen. Controls for the experiment were (1) a reaction mixture in which BSB substituted for unlabeled antigen and (2) BSB and normM serum substituted for unlabeled antigen and antiserum.

pH Optima and Heat Inactivation o/Neuraminidase Virus samples were incubated with fetuin at different pH values and assayed for neurarninidase activity as described. Virus was incubated at 45 ° C in a water bath. Samples were removed at various times and assayed for neuraminidase activity.

Results

Purification o/ Virus A d s o r p t i o n of virus to RBC, followed b y its elution a n d s e d i m e n t a t i o n , resulted in a 50 per cent recovery of virus H A a n d 54.8-fold purification on the basis of H A / m g p r o t e i n (Table 1). A n 81.5-fold purification was achieved b y s e d i m e n t a t i n g virus t h r o u g h a sucrose gradient. Table 1. Puri/ication o/ Swine Influenza Virusa

Sample

Total volume in ml

Total Percent Total HA recovery protein ( × 104) (I-IA) (rag)

Percent HA/rag recovery of (protein) protein

Allantoie fluid

2000

12.8

100

Eluted virus

t00

2000 228

300

7.6

60

Sedimented eluate (partially purified)

4

6.4

50

Sucrose gradient purified

1.5

1.2

11.4

64 336.8

Purifica~ tion (fold) 1 5.2

18.2

0.9

3508

54.8

2.3

0.1

5217

81.5

9.2

Virus was purified from infected allantoic fluid by absorption and elution on RBC. The etuted virus was further purified by sedirnenting through 10--40 per cent linear sucrose gradient, at 35,000 g for 45 minutes, and concentrated by centrifugation a t 50,000 g for 3 hours. HA titer and protein content of each sample were determined to calculate specific activity (HA units/mg of protein).

Serological Tests Table 2 shows t h a t SW/73 a n d S W / 3 i a n t i g e n s cross-reacted in the H I t e s t with antisera. A n t i - S W / 7 3 d e m o n s t r a t e d stronger cross-reaction b e t w e e n t h e two antigens t h a n did SW/31 a n t i s e r u m .

248

D.M. SATIn!,L. S. RODXE¥, and H. C. MINOOJtA:

Table 2. Serological Cross-Reactions between A/Swine/Shope 15/31, and A/Swine~ Wisconsin~I~73 in the Hemagglutination Inhibition .Test Antigens Antiserum

Alswine/Wisconsin/1/73

Alswine/Shope 15131

A/swine/Wisconsin/I/73 A/swine/Shope 15/31

1024a 640

512 3072

Ftemagglutination inhibition titers, expressed as reciprocals of serum dilution. Figures i a and b demonstrate a close relationship of SW/73 and SW/31 viruses in N I test. Nevertheless, at 1:40 dilution of anti-SW/73 inhibition of SW/73 neuraminidase activity was 30 per ccitt greater than t h a t of SWt31. These results, together with the profile of curves in N I test, indicated antigenic differences between the two viruses. Auti-SW/31, however, did not detect any significant difference between the two enzymes (Fig. 1 b). $0 I

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Antigenic Comparison of Swine Influenza Virus Isolates

249

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