Subunits of Hemagglutinating Virus of Japan

JOURNAL OF VIROLOGY, OCt. 1970, p. 492-499 Vol. 6, No. 4 Copyright 1970 American Society for Microbiology Printed in U.S.A. Isolation of Hemagglutinin and Neuraminidase Subunits of Hemagglutinating Virus of Japan KOICHIRO MAENO, TETSUYA YOSHIDA, MASAO IINUMA, YOSHIYUKI NAGAI, TOSHISADA MATSUMOTO, AND JUNPEI ASAI Department of Virology, Cancer Research Institute, and Department of Pathology, Nagoya University School of Medicine, Nagoya, Japan Received for publication 15 June 1970 When purified hemagglutinating virus of Japan (HVJ) was treated with trypsin, two major surface antigens were released from the virus. The "hemagglutinin" subunits obtained by this method were reactive with homologous hemagglutination-inhibition antibody and could be detected by an antibody-blocking test. They adsorbed to but did not agglutinate red cells and thus appeared to be "monovalent." The neuraminidase subunits were obtained in fully active form and did not adsorb to red cells. This finding suggests that these two activities of HVJ are associated with different subunits of the virus particle. The hemagglutinin and neuraminidase subunits could be partially separated by zonal rate centrifugation or gel filtration on Sephadex G-200. The molecular weights estimated for these subunits were approximately 124,000 and 114,000, respectively. After treatment with trypsin, virusassociated hemagglutinin and neuraminidase activities were both reduced significantly. The electron micrographs of such trypsinized virus particles showed complete or partial loss of surface projections. These results suggested that the subunits obtained by this method seemed to be those projections liberated from the virus by the action of trypsin. Extensive studies have been carried out on the recombinant influenza viruses possessing hemagglutinin subunit from one parent and neuraminidase from the other, antigenically unrelated parent (6, 7). These recombinants have proven to be very useful in the preparation of antibodies against either the hemagglutinin or the enzyme subunits (13, 14). In contrast to influenza virus, many attempts to demonstrate recombination with paramyxoviruses have been as yet unsuccessful. It therefore seems important to isolate hemagglutinin and neuraminidase subunits from paramyxoviruses, not only to characterize these subunits but also to obtain specific antibodies against them. Recently, Laver and Valentine reported that treatment of influenza virus with sodium dodecyl sulfate (SDS) resulted in the isolation of hemagglutinin and neuraminidase in biologically active form (8). Accordingly, we used this method in an attempt to isolate similar subunits from hemagglutinating virus of Japan (HVJ; Sendai virus), but this was unsuccessful because such treatment was always accompanied by concomitant destruction of both biological activities of the virus. During further investigation, we found that digestion of HVJ with trypsin leads to a successful 492 isolation of hemagglutinin and neuraminidase subunits from the virus. In this paper, evidence will be presented which suggests that these activities are associated with the surface projections of the HVJ particle. Separation of these subunits by zonal rate centrifugation and by the gel-filtration techniques will also be described. MATERIALS AND METHODS Virus and antiserum. The Nagoya 1-60 strain of HVJ grown in the allantoic cavity of 11-day-old chick embryos was purified by differential centrifugation and sedimentation through a linear 10 to 40% sucrose gradient in phosphate-buffered saline (PBS) at 15,000 rev/min for 30 min. Twelve-drops fractions were collected from the bottom of the tube and assayed for their biological activity. Figure 1 shows the distribution of hemagglutinin, neuraminidase, and infectivity. The peak fractions were pooled and used as purified HVJ throughout this study. Anti-HVJ serum was obtained from rabbits immunized with purified HVJ. Specific antibody against viral surface antigens was prepared by dissociation of virus-antibody complex in 0.1 M glycine-hci buffer at ph 2.7. Hemagglutination titrations. Serial twofold dilutions of virus in 0.25-ml volumes of PBS were made in plastic trays. Chicken erythrocytes were added as a 0.025-ml drop of a 5% suspension. Titers were read

VOL. 6, 1970 HEMAGGLUTINATING VIRUS OF JAPAN 493 C4 or 1% O 1 5 10 15 20 FRACTION Log1o E I-D5on0l -1 10 FIG. 1. Purification of HVJ by sucrose gradient centrifugation. Virus was collected from infected allantoic fluids by differential centrifugation. The concentrated virus was layered onto a linear 10 to 40%o sucrose gradient in PBS and centrifuged at 15,000 revl miii for 30 min in an S W 25 Spinco rotor. Fractions 12 to 14 were pooled, dialyzed against PBS at 4 C, and used as purified HVJ throughout this study. Symbols: 0, hemagglutination (HA) unzits; X, inifectivity (EID5o); *, neuraminidase activity (OD549). by the pattern method after 35 min at room temperature. Hemagglutination-inhibition (HI) tests. Serial twofold dilutions of antibody in 0.2-ml volumes were made in plastic trays. To each dilution, 0.1 ml of 4 hemagglutination units of virus was added and incubated for 1 hr at room temperature before the addition of chicken erythrocytes. The highest dilution of antibody which caused complete inhibition of hemagglutination was regarded to contain HI unit of antibody. Antibody-blocking tests for hemagglutination. Four HI units of antibody (in 0.1 ml) was mixed with serial twofold dilutions of test material (in 0.2 ml) and allowed to react together for hr at room temperature before the addition of 4 hemagglutination units of whole virus (in 0.1 ml). The mixtures were incubated for an additional hr and then 0.025 ml of chicken erythrocytes was added. Hemagglutination indicated the presence of nonhemagglutinating antigen. The titer of HI antibody-blocking activity was expressed as the highest dilution of the test material showing hemagglutination. Neuraminidase assay. Neuraminidase assay was performed with a sialyl lactose substrate by a modification of the thiobarbituric acid method of Warren (7, 9). A 0.2-ml volume of test materials diluted with saline was added to 0.1 ml of substrate and adjusted to ph 5.0 with 0.1 ml of 0.4 M sodium acetate buffer. After 30 min of incubation in a water bath at 37 C, 0.2 ml of the reaction mixture was used for the assay of free N-acetylneuraminic acid. Optical density was read at 549 nm (ODM9) against a blank tube containing sialyl lactose plus saline. Enzyme activity was measured by OD readings taken from the part of the slope where they vary linearly with the enzyme concentration. Sialyl lactose was prepared by the method of French and Ada (3). Infectivity titrations. Infectivity titrations of HVJ were carried out by intra-allantoic inoculation of four or five chick embryos with 0.1 ml of each of 10-fold serial dilutions of test materials. Allantoic fluids were tested for hemagglutination after 3 days of incubation at 36 C. The 50%c infectivity end points (EID.O) were calculated by the method of Reed and Muench. Treatment of HVJ with trypsin. Purified HVJ was mixed with Difco trypsin (1:250) at a final concentration of 1.0 mg/ml in 0.1 M phosphate buffer (ph 7.6) and incubated in a water bath at 37 C. At various times of incubation, soybean trypsin inhibitor was added at a final concentration of 1.5 mg/ml and the mixtures were chilled in an ice bath to stop the action of the enzyme. After centrifugation at 35,000 rev/min for 1 hr, the upper one-half of the supernatant fluids was harvested. The sediments were also harvested after resuspending to original volumes in PBS. Zonal rate centrifugation. A linear 5 to 20%7c sucrose gradient in PBS (ph 7.2) was used. Samples were applied onto the gradient and centrifuged at 35,000 rev/min for 12 hr in an SW 39 rotor. Fractions collected from the bottom of the tube were dialyzed against PBS to remove sucrose and assayed for biological activity. Gel filtration on Sephadex G-200. A 0.5-ml volume of test material was applied to the Sephadex column (1.6 by 84.0 cm) equilibrated with 0.025 M phosphate buffer (ph 7.2) containing 0.1 M NaCl. The column was eluted with the same buffer, and 2.5-ml fractions were collected and assayed for biological activity. The void volume of the column was determined by using a Dextran Blue marker. Molecular weight of the test material was estimated by using human gamma globulin (molecular weight, 160,000), ovalbumin (molecular weight, 45,000), and cytochrome c (molecular weight, 12,400) as marker proteins. Electron microscopy. The negative staining method was employed. Drops of virus or subunit preparations were placed on carbon-coated grids and excess fluids were removed with a filter paper. Each grid was then covered with a drop of 1%7o potassium phosphotungstate (ph 7.4), and excess fluid was absorbed with a filter paper. After the preparations were left to dry in the air, they were examined in a Hitachi HU-1 I D electron microscope. RESULTS Effect of trypsin on hemagglutinin and neuraminidase activities of HVJ. It has been reported that after removal of most of the neuraminidase from influenza virus by treatment with trypsin, the residual virus retains full hemagglutination

494 MAENO ET AL. J. VIROL. activity (11). If a similar result could be obtained with HVJ by treatment with trypsin, the isolated neuraminidase subunits and the residual virus would be useful in the preparation of antibodies against each of two major surface antigens of HVJ. Experiments were therefore undertaken to examine in detail the effect of trypsin on the hemagglutinin and neuraminidase activities of HVJ. Purified preparations of HVJ were mixed with trypsin and incubated at 37 C as described above. At various times of incubation, soybean trypsin inhibitor was added and the reaction mixtures were chilled in an ice bath to stop the action of the enzyme. After centrifugation at 35,000 rev/ min for 1 hr, both the upper one-half of the supernatants and the sediments resuspended to original volumes in PBS were harvested and assayed for their hemagglutinin and neuraminidase activities (Fig. 2). As shown in Fig. 2, hemagglutinin activity associated with sediments decreased gradually with incubation time. Only one-fourth of the original hemagglutinin could be recovered in the sediment after 1 hr of incubation with trypsin. None of the supernatant fractions showed any hemagglutinin activity. This result suggested the possibility that the hemagglutinin of HVJ is readily destroyed by the action of trypsin. Alternatively, the hemagglutinin subunits of HVJ might be released from the virus with concomitant loss of hemagglutinating activity. It IC (C z 6400 0 3200 O 1600 X IX Soo I- 400 200 --. X-8- -- 0 30 60 120 INCUBATION TIME (Imin_ M) FIG. 2. Effect of trypsin on hemagglutinin activity of HVJ. Purified HVJ was treated with trypsin (1.0 mg/ml). At the end of the indicated incubation periods, the reaction was stopped by the addition of soybean trypsin inhibitor (1.5 mg/ml) and chilling. The mixture was centrifuged at 35,000 rev/min for I hr, and the supernatant and sediment (resuspended to original volume in PBS) were assayed for hemagglutination (HA). Symbols: 0, total HA activity; X, virus-associated HA activity; 0, HA activity in the supernatant; O, HA activity of untreated virus (control). was reasonable to assume that the subunits separated from virus were "monovalent" so that they did not cause agglutination of red cells. If, however, these subunits still retained the capacity to combine with homologous antibody, an antibodyblocking test might reveal the presence of such subunits in the supernatant fluids. To test this possibility, experiments were carried out (Table 1). It was found that agglutination of red cells by virus occurred in the presence of a sufficient amount of antiviral antibody to inhibit the agglutination, if the antibody was allowed to react with the supernatant fluids previously. This result suggested that, by the treatment of HVJ with trypsin, some agent or subunit antigenically similar to hemagglutinin but without the capacity to agglutinate red cells might be released into the supernatant fluids. Results of neuraminidase assay are shown in Fig. 3. It was found that the neuraminidase of HVJ could also be separated from the virus in soluble form. As can be seen in Fig. 3, more than 50% of the virus-associated neuraminidase activity was released from virus in 30 min, and thereafter the enzyme activity in the supernatant fractions decreased gradually. Adsorption of the HI antibody-blocking agent to red cells. From the results of experiments described in the preceding section, it was assumed that, by treatment of HVJ with trypsin, two kinds of viral subunit were released from the virus, one of which was associated with neuraminidase activity and the other of which was associated with HI antibody-blocking activity. However, the possibility existed that both of these two activities were associated with the same subunit. To test this possibility, adsorption capacity of each of TABLE 1. Effect of trypsin on liberation of HI antibody-blocking antigen (hemagglutinin) from HVJ Time of treatment (min) rhi antibody-blocking activitya Total Supernatant Pellet 0 640 <20 640 30 640 1,280 160 60 640 640 80 120 320 640 40 240 160 320 20 a Purified preparations of HVJ were treated with trypsin for various times indicated above and assayed for HI antibody-blocking activity before and after centrifugation at 35,000 rev/min for 1 hr. In this test, hemagglutination indicated the presence of antigen, and the end point of the test was recorded as for standard hemagglutination titrations. Values expressed as hemagglutination units per 0.2 ml.

VOL. 6, 1970 HEMAGGLUTINATING VIRUS OF JAPAN 495 30 60 120 20 INCUATION TIME FIG. 3. Kinletics of release of neuraminidase by treatment withl trypsin. For th2e methlod of treatmenlt of HVJ Withl trypsin, see the legenld to Fig. 2. Symbols: 0, total enlzyme activity; *, enzyme activity in the supernlatanlt; X, virus-associated entzyme aclivity. these activities to red cells was examined (Table 2). Purified preparation of HVJ was mixed with trypsin and incubated at 37 C for 45 min to allow maximum release of neuraminidase and HI antibody-blocking activity with a minimum of inactivation. After addition of soybean trypsin inhibitor to stop the action of the enzyme, virus was sedimented by centrifugation and the supernatant was harvested as described above. A mixture of 1 ml of the supernatant with 0.2 ml of packed red cells was allowed to stand in an ice bath for 1 hr, after which the cells were removed by centrifugation at low speed and the resulting supernatant was tested for HI antibody-blocking activity. As shown in Table 2, the HI antibody-blocking agent adsorbed onto red cells, whereas the neuraminidase did not. It therefore seems likely that these two activities are associated with different subunits. For a comparison, ether-disrupted HVJ was tested in a similar way. After absorption of the ether-disrupted preparation of HVJ with red cells, we could not detect any remaining hemagglutinin or neuraminidase activity in the supernatant. This result suggested that disruption of HVJ with ether would result in a release of a viral subunit which possessed both hemagglutinin and neuraminidase activities. Electron microscopic examination. As described above, when HVJ was treated with trypsin, the residual virus appeared to have lost most of the hemagglutinin and neuraminidase activities. The morphological appearence of the virus was therefore examined. After treatment with trypsin for 45 mw, virus was purified by two cycles of differential centrifugation. The virus was stained negatively with 1V potassium phosphotungstate and examined in a Hitachi HU-1 1 D electron microscope. The electron micrographs sh owed complete or partial elimination of projections from the viral surface (Fig. 4A). The morphological appearance of the subunits was also examined. The subunits were purified by chromatography on a Sephadex G-200 column and concentrated by dialyzing against polyethylene glycol as described below. Although the electron microscopic examination revealed numerous small particles which appeared to be almost the same size as the spikes of HVJ, identification of these particles with the neuraminidase or hemagglutinin subunit needs further examination (Fig. 4A, inset). Separation of hemagglutinin and neuraminidase. From the results of experiments described in the preceding section, it is suggested that the HI antibody-blocking activity is actually associated with the hemagglutinin subunits released from virus. The hemagglutinin and neuraminidase subunits of trypsin-treated HVJ were separated on a sucrose gradient. Purified preparation of HVJ was treated with trypsin for 45 min and centrifuged at 35,000 rev/min for 1 hr to remove virus particles. The supematant fluid was concentrated fivefold by dialyzing against polyethylene glycol at 4 C. A 0.1-ml volume of the concentrated material was applied onto a linear 5 to 20% sucrose gradient. The distribution of hemagglutinin and neuraminidase activities after centrifugation at 35,000 rev/min for 12 hr was illustrated in Fig. 5. As can be seen in the figure, each of these activities sedimented as a homogeneous peak. Al- TABLE 2. Adsorption of HI anttibody-blocking anztigent (hemagglutininz) anid nieutraminiidase sutbuniits to red blood cells Activities before and after absorption with red cellsa Before After Exptl Antibody- -Neuramini- Antibody- -Neurablocking I dase blocking min idase activity activity activity activity (HAU/0.2 ml)b (7c) (HAU/0.2 ml) (%7) 1 512 100 64 110 2 512 100) 128 160 3c 32 100 <1 5 a Absorbed with guinea pig erythrocytes. bhau, hemagglutination units. c Ether-disrupted virus. Purified preparation of HVJ was mixed with an equal volume of peroxide-free ether. After removal of the ether by nitrogen bubbling, the sample was centrifuged at 20,000 rev/min for 1 hr, and the supernatant was harvested and examined.

FIG. 4. Electroni micrographis of HVJ stained with 1%> potassium phosphotunlgstate. Magnification, X 200,000. Bar represents 50 flm. (A) HVJ after treatment with trypsini showing the removal of surface projections. Inset: the particles collected from the sutperitatalnt of trypsini-treated HVJ by fractionation through colutmni chromatograplhy ont Sephadex G-200. (B) Untreated viruts. 496

VOL. 6, 1970 HEMAGGLUTINATING VIRUS OF JAPAN 497 5 10 1S FRACTION FiG. 5. Sedimentation of the hemagglutinin and neuraminidase subunits of HVJ. Purified HVJ was treated with trypsin for 45 min, and the mixture was centrifuged at 35,000 rev/min for I hr to remove virus pariicles. The supernatant was harvested and concentrated fivefold by dialyzing against polyethylene glycol at 4 C. A 0.1-ml amount was applied onto a linear 5 to 20% sucrose gradient and centrifuged at 35,000 revl min for 12 hr in an SW 39 rotor. Samples were collected from the bottom ofthe gradient, diluted 10-fold in PBS, and dialyzed against PBS to remove sucrose. Hemagglutinin and neuraminidase activities were estimated as described. Hemagglutinin could only be detected by an antibody-blocking assay. Symbols: 0, antibody-blocking activity [hemagglutination (HA) units]; *, neuraminidase activity (OD549). though these peaks were adjacent, it was clear that the hemagglutinin sedimented at a faster rate than neuraminidase. Separation of subunits by column chromatography on Sephadex G-200 was also attempted. After filtration through the column, each hemagglutinin and neuraminidase activity was also recovered as a homogeneous peak in a position intermediate between gamma globulin and ovalbumin peaks (Fig. 6). The molecular weights estimated for hemagglutinin and neuraminidase subunits were approximately 124,000 (+:4,000) and 114,000 (43,000), respectively. DISCUSSION The early observation that particles of myxoviruses possess a large number of projections on their surface has stimulated much interest in the relationship between these projections and the biological activities known to be associated with the viral surface (4). Mayron et al. (10) first reported that treatment of influenza virus with I trypsin resulted in the separation of most of the z neuraminidase activity from the virus. Noll and 9 co-workers (11) confirmed this result and sug- '~r gested further that the surface projections of in-.o *t fluenza virus were responsible for the hemag- o glutinin activity and that the neuraminidase z molecule might be situated between these hemaga glutinin spikes. They found that, after treatment F3 with trypsin, the residual virus retained full hemagglutination activity and did not appear to have lost any of its surface projections. On the other hand, Laver and Valentine (8) have recently reported that the biologically active hemagglutinin and neuraminidase subunits could be successfully isolated by disruption of influenza virus with SDS. Their results suggest that the spikes seen on the surface of influenza virus are of two OD549 2.000 l,soo_ 1000_- 0OS0O_ 5 10 15 20 25 30 FRACT ION M.W. -4.' 'Ak 0l 2 p 0-0 9 - '-- *-0_ FIG. 6. Chromatography on Sephadex G-200 of hemagglutinin and neuiraminidase subunits of HVJ. A 0.5-ml amount of test material (see the legend to Fig. 5) was applied to a Sephadex G-200 column equilibrated with PBS (ph 7.2). The column was eluted with PBS (ph 7.2), and 2.5-ml samples were collected and assayed for neuraminidase activity (0) and hemagglutinin activity (0). The hemagglutinin could be detected by an antibody-blocking test. Calibration of the column (A) was carried out with human gamma globulin (molecular weight, 160,000), ovalbumin (molecular weight, 45,000), and cytochrome c (molecular weight, 12,400). The column dimensions were 84 by 1.6 cm; temperature was maintained at 4 C. 5.0 32 16 (2 z 0 0 I 0 S -

498 MAENO ET AL. J. VIROL. morphologically distinct kinds, one of which is associated with the neuraminidase activity, and the other of which is associated with the hemagglutinin. Our studies described in the present communication indicate that, when HVJ was treated with trypsin, both the hemagglutinin and neuraminidase subunits were released from the virus, and, as a result, the residual virus was deprived of hemagglutinating and enzymatic activity. Electron micrographs of the trypsinized HVJ show complete or partial loss of surface projections. Similar morphological changes have been observed with influenza viruses and vesicular stomatitis virus treated with various proteases (1, 2, 5). The approximate estimates of molecular weight of the isolated hemagglutinin and neuraminidase subunits obtained from the sieving characteristics through Sephadex G-200 were in the range of 110,000 to 130,000. The volume of surface projection of HVJ calculated from its morphological appearance and the average protein specific volume (0.73 cm3/g) indicated a molecular weight for the projection of around 110,000. It has been proposed that the projection of HVJ has a spherical head 6.0 nm in diameter and a tail of 8.0 nm in length and 2.0 nm in width. The total length of the projection is not known, because the end of the tail appears to be embedded in the membrane of viral envelope (Y. Amano, K. Tada, and N. Ishida, unpublished data). The similarity in their size suggested rather strongly that these projections could be liberated from the virus without significant changes in their size and that they were the structural units responsible for the hemagglutinin and neuraminidase activities. Since trypsin splits proteins specifically at the carboxyl groups of arginine and lysine residues, the size of the subunit obtained by this enzyme would depend upon the location of the trypsin-sensitive site of the molecule. As has been described, in our attempts to separate the hemagglutinin and neuraminidase subunits by chromatography on Sephadex G-200 or sedimentation through sucrose gradient, each of these activities was detected as a homogeneous peak. This result suggested that trypsin may split the polypeptide chain at a definite site on the projection and thus result in release of subunits possessing uniform size. Thus, our findings seemed to be in good agreement with the concept proposed by Laver and Valentine (8) and by Webster and Darlington (12). It seems likely that the spikes on the surface of HVJ are of two kinds, one of which is associated with neuraminidase activity, and the other of which is associated with the hemagglutinin. However, the properties of hemagglutinin subunits of HVJ obtained by this method differed from those of influenza virus subunits obtained by the SDS or Tween 20 method. The hemagglutinin subunits of HVJ were reactive with homologous HI-antibody and adsorbed to red cells, but they did not have the capacity to agglutinate them. It is thought that this was because the subunits were monovalent and attached to the cell receptors at one end only and because they could not form polymers or aggregates under the experimental conditions employed. Nevertheless, it is reasonable to expect that the hemagglutinin subunits could induce formation of antibody when introduced into animals. The specific antibodies against the hemagglutinin and neuraminidase subunits would be useful tools in the study of the intracellular development of these viral components independently. ACKNOWLEDGMENTS The authors thank 1. Nagata, Y. Kimura, and N. Ogas.awara for their helpful discussions. 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