Isolation and Characterization of Defective. Disease Virus

Transcription

1 Microbiol. Immunol. Vol. 22 (12), , 1978 Isolation and Characterization of Defective Interfering Particle of Newcastle Disease Virus Akitoshi MAEDA,1 Yasuo SUZUKI, and Makoto MATSUMOTO Department of Biochemistry, Shizuoka College of Pharmacy, Shizuoka and Central Laboratories, Kyorin Pharmaceutical Co. Ltd., Tochigi (Received for publication, January 31, 1978) Abstract Newcastle disease virus grown in embryonated eggs was separated and purified by sucrose density gradient centrifugation into two distinct types of particles, B and T, the former being normal virus particles with high activities of hemagglutination, hemolysis, neuraminidase and infectivity, the latter being non-infectious virus particles with low activities of hemolysis and neuraminidase but high hemagglutination activity. B and T particles were shown to share a common antigen by immunodiffusion test. T particles were deficient in viral RNA, since they contained only 13s RNA in a small amount, whereas B particles possessed a large amount of 57s RNA and a small amount of 13s RNA. T particles interfered with the multiplication of normal Newcastle disease virus in primary cultures of chick embryo cells. Newcastle disease virus (NDV) produced in embryonated eggs has been reported to consist of two distinct types of particles, i.e. infectious hemagglutinating (IH) and non-infectious hemagglutinating (NIH) particles (3, 7, 13, 14). In contrast to IH particles which are normal and fully active virus, NIH particles are smaller (3), non-hemolytic (1), deficient in viral RNA (13) and do not show interference of von Magnus type (3, 14). In the present study, NIH particles were separated and purified from NDV produced in chick embryos, and their biological properties were investigated. NIH particles were shown to be defective virus particles, and able to interfere with the replication of normal NDV, indicating that they are "defective interfering particles" as defined by Huang and Baltimore (4). MATERIALS AND METHODS Virus. A plaque-purified line of the Miyadera strain of NDV was kindly supplied by Dr. H. Shibuta, Institute of Medical Science, Tokyo University. About 104 PFU of virus was innoculated into the allantoic cavity of 11-day-old eggs, which were then incubated for 48 hr at 36 C. After incubation at 4 C overnight, allantoic fluid was harvested, centrifuged to remove cells and coarse debris, and stored at 1 Present address: Central Laboratories, Kyorin Pharmaceutical Co. Ltd., Nogi-machi, Tochigi, Japan. 775

2 776 A. MAEDA ET AL Fractionation and purification of virus. Infectious allantoic fluid was centrifuged at 30,300 ~ g for 90 min or at 63,600 ~ g for 60 min at 4 C, and the precipitates were suspended in phosphate buffered saline (PBS). The virus suspension was centrifuged again in the same way, and the precipitates were suspended in an appropriate volume of PBS to obtain a concentrated virus suspension. The suspension was then centri- fuged in a gradient of sucrose in PBS (10-40%, w/v) at 40,700 ~g for 45 min, or at 73,450 ~ g for 30 min at 4 C, and fractions were collected from the bottom of the centrifuge tube. Hemagglutination activity (HA) and hemolytic activity (HL) of each fraction were estimated as described later, and fractions with high HA and HL activities near the bottom were pooled to give fraction B-1. Fractions with high HA and low HL activity found near the top of the tube were combined to give fraction T-1. B-1 and T-1 fractions were dialyzed against PBS at 4 C for 18 hr, concentrated by centrifugation and further subjected twice to the sucrose density gradient centrifugation. The two fractions obtained by the second sucrose density gradient centrifugation were designated as B-2 and T-2 and those from the third one as B-3 and T-3, respectively. By this purification procedure, it was found that the peaks of HA and HL overlapped finally in a single peak in both fractions. Removing subindices from B-3 and T-3, the final purified virus suspensions were designated as B and T. Preparation of 3H-uridine-labeled virus. Eleven-day-old eggs were injected with 104 PFU/egg of the seed virus and 100 Đci/egg of 5-3H-uridine (Daiichi Chem. Co. Tokyo) into the allantoic cavity, incubated for 48 hr at 36 C, and allantoic fluid was harvested. Virus was concentrated, fractionated and purified in the same manner as described above. Analysis of viral RNA. The purified virus B and T, labeled with 3H-uridine, were solubilized in TE buffer (5 mm Tris-HC1 buffer-1 mm EDTA) containing 1% SDS at 60 C for 30 sec, and were centrifuged in a density gradient of 5-20% (w/v) sucrose-0.5% SDS in TE buffer at 53,100 ~ g for 18 hr (5). Each fraction collected from the tube bottom was analyzed for the radioactivity of acid-insoluble fraction by a liquid-scintillation counter (Aloka, LSC 602). Ribosomal RNA prepared from E. coli, K-12 MO (Miles Laboratory, Inc., Illinois, U.S.A.) was used as the standard RNA for the centrifugational analysis. Hemagglutination (HA) activity. HA titer was determined by Salk's pattern method (15), and the titer was expressed by the highest dilution of virus suspension giving the minimum agglutination. Hemolytic (HL) activity. HL activity was estimated by the method of Kohn (6) using 2% chicken erythrocyte suspension, and was expressed by the absorbancy at 540 nm shown by hemoglobin released from hemolyzed cells. Infectivity. Infectivity was estimated by the plaque count method of Maeno et al (9). The titer was expressed in plaque-forming units (PFU). Neuraminidase activity. This was determined using fetuin (Grand Island Biochemical Co., Berkeley, California, U.S.A.) as a substrate (10). Protein. This was measured by the method of Lowry et al (8) using bovine serum albumun as a standard.

3 DEFECTIVE INTERFERING PARTICLE OF NDV 777 Antiserum. A rabbit received 4 weekly intravenous doses of concentrated NDV suspension. Ten days after the last injection, serum was obtained, and its HAinhibiting antibody titer was estimated to be 1: 1,280. Immunodiffusion test. This was carried out according to the usual method on a plate of 1% agar (Difco Laboratories, Detroit, Michigan, U.S.A.) (11). Electron microscopy. Virus particles were examined by the negative staining technique with 4% uranyl acetate, in an electron-microscope, Hitachi HU-11B. Cell cultures. Primary cultures of chicken embryo cells were grown in Eagle's MEM (minimum essential medium) containing 5% bovine serum (BS), 10% tryptose phosphate broth (TPB) (Difco Lab.). After virus inoculation the cultures were maintained in Eagle's MEM containing 2% BS and 5% TPB. RESULTS Fractionation and Purification of NDV NDV was fractionated into two fractions by sucrose density gradient centrifuga- Fig. 1. Fractionation of egggrown NDV by sucrose density gradient centrifugation. One ml of concentrated virus was layered on 30 ml of linear 10-40% (w/v) sucrose gradient in PBS and centrifuged for 45 min at 40,700 ~ g. Fractions of 2 ml each were collected from the tube bottom and tested for HA Table 1, Purification of NDV (B, T)

4 778 A. MAEDA ET AL Fig. 2. Purification of B and T by sucrose density gradient centrifugation. B-1 and T-1 shown in Fig. 1 were recentrifuged individually twice more in the same way as described tion, B-1 with high HL and HA activities, and T-1 with low HL activity and high HA activity (Fig. 1). The B-1 and T-1 fractions thus obtained were further subjected to two cycles of dialysis, concentration, and sucrose density gradient centrifugation. The virus purification and percentage of recovery were monitored by HA and protein determinations (Table 1). Viral specific activities, defined as HAU per milligram of protein, reflected 58-fold and 123-fold purification of the final purified preparations, B and T, respectively. The purity of these preparations was also supported by a sharp peak demonstrated by sucrose density gradient centrifugation (Fig. 2). Biological Activities of B and T Comparison of various biological activities between B and T preparations was made on the basis of equal HA activity (Table 2). T particles showed lower relative activities than B particles. HL, neuraminidase and infectivity of T particles were about 1/4, 1/6 and 10-7 of those of B particles, respectively. Antigenicity of B and T Antigenic comparison between B and T preparations were made by the immunodiffusion test. test. B and T particles solubilized with SDS were shown to form precipi-

5 DEFECTIVE INTERFERING Table Fig Biological Immunodiffusion PARTICLE activities OF NDV 779 of B and T test of purified B and T against anti-ndv serum. Antigen (B and T) and anti-ndv serum were treated with 1% SDS before being added to the agar plate. Rabbit antindv serum was placed in the center well. Precipitation lines were observed after 48 hr-incubation at room temperature. tation lines with anti-ndv rabbit serum fusing each other to give a single line, indicating that B and T particles possess at least one antigen in common (Fig. 3). MorphologicalObservationof B and T Electron microscopic observation has revealed that B and T preparations contain spherical particles having spikes on their surface. B particles were larger than T particles, the former measuring nm in diameter, and the latter nm across (Fig. 4). Analysisof RNA of B and T Analysis of 3H-uridine-labeled viral RNA by sucrose density gradient centrifugation gave two peaks corresponding to 57s and 13s with B particles. On the other hand, T particles were found to contain only 13s RNA, showing that T particles were defective in RNA composition (Fig. 5).

6 780 A. MAEDA ET AL B T Fig. 4. Electron micrograph of B and T. Particles were negatively stained with 4% uranyl acetate (scale 200 nm). Interferencewith Multiplication of B by T T particles interfered with multiplication of B virus in primary monolayer cultures of chick embryo fibroblasts (Table 3). The interference was high even at low m.o.i. of T particles.

7 DEFECTIVE INTERFERING PARTICLE OF NDV 781 Fig. 5. Sucrose density gradient centrifugation of 3H-RNAs of B and T. B and T were dissociated with 1% SDS for 30 sec at 60 C and centrifuged indivi- dually at 53,100 ~ g for 18 hr on linear 5-20% (w/v) sucrose gradient in TE buffer. Each fraction was collected from the bottom of the tube and analyzed for the radioactivity of trichloroacetic acid-insoluble fraction. Table 3. Interference with the growth of B in chick embryo fibroblasts by T Monolayer cultures of chick embryo fibroblasts were infected with B alone or B and T at a multiplicity of infection as indicated in the Table. After incubation for 40 hr, the infectivity of the culture fluid was estimated. a) 24 hr b) 36 hr c) 48 hr Fig. 6. Production of T in embryonated eggs infected with normal B virus. Virus was harvested from embryonated eggs 24, 36 and 48 hr after infection with 4.1 ~ 105 PFU/egg of purified B. Virus was concentrated by centrifuga-

8 782 A. MAEDA ET AL Fig. 7. Centrifugal profile of HA and HL activities of NDV released in vitro from infected chorioallantoic mem- branes. Chorioallantoic membranes were taken from embryonated eggs 24 hr after infection with 4.1 ~ 105 PFU/egg of purified B, washed twice with PBS and were cultivated in vitro as described in the text. The virus released into the medium during 24 hr-incubation in vitro was concentrated by centrifugation and fractionated in the same way as de- Production of T Particles in the Embryonated Eggs Infected with B Virus Observation was made periodically on the production of T particles in embryonated eggs infected with normal B virus (Fig.6). Twenty four hr after the innoculation with 4.1 ~ 105 PFU/egg of purified B virus (B-3), no T particles were demonstrated in the allantoic fluid, whereas T particles were found at 36 and 48 hr. A similar result was obtained with 10-fold greater innoculum of B virus than that of the above described experiment. In another experiment virus-infected chorioallantoic membranes were taken out from embryonated eggs 24 hr after innoculation with 4.1 ~ 105 PFU/egg of B, washed twice with PBS and cultivated at 37 C in MEM containing 2% TPB for 24 hr. At the end of cultivation the culture fluid contained T-like particles with high HA and low HL activities (Fig.7).

9 DEFECTIVE INTERFERING PARTICLE OF NDV 783 DISCUSSION In the present study, two distinct types of viral particles, B and T, were separated from NDV grown in the embryonated eggs, and purified by repeated centrifugation in the sucrose density gradient. The results of test for biological activities indicated. B particles to be normal active virus and T particles to be noninfectious hemagglutinating (NIH) viral particles reported previously by several workers (3, 7, 13, 14). Although T particles were morphologically similar to B particles, T particles were significantly smaller than B particles as reported previously with NIH particles (3). T particles were deficient in hemolytic activity as compared with normal B particles, confirming the previous report (1). T particles were found to contain only 13s RNA in a small amount, whereas B particles had a large amount of 57s RNA and a small amount of 13s RNA, indicating T particles to be deficient in viral RNA. NDV has been reported to contain 57s RNA (2), but the presence of 13s RNA has not yet been reported. Furthermore, T particles were also deficient in neuraminidase activity. On the other hand, the immunodiffusion test demonstrated T particles to be antigenically related to normal B particles. Moreover, T particles interfered with virus replication in chick embryo cell cultures infected with B particles. These results indicate T particles to be "defective interfering" (DI) particles as defined by Huang and Baltimore (4). Recently, Roman and Simon (12) reported that NDV gave rise to DI particles by serial undiluted passages in chick embryo fibroblasts, but not in embryonated eggs. In the present study, however, T (DI) particles were shown to increase significantly even by one diluted passage (about PFU/egg) in embryonated eggs. These results indicate that the mode of interference with T particles may not be von Magnus type. As Huang and Baltimore (4) suggested, DI particles may play a role in the course of viral infections. Studies on DI particles of NDV may therefore contribute much to gain an insight into the pathogenesis of NDV infection. Much more studies are needed to elucidate the mechanism of the production of DI particles in relation to the viral synthesis. The molecular basis of the biological activities of DI particles should also be solved. REFERENCES 1) Clavell, L.A., and Bratt, M.A Hemolytic interaction of NDV and chicken erythrocytes. II Determining factors. Appl. Microbiol. 23: ) Duesberg, P.H., and Robinson, W.S Isolation of the nucleic acids of Newcastle disease virus (NDV). Proc. Natl. Acad. Sci. U.S. 54: ) Granoff, A Noninfectious formes of Newcastle disease and influenza virus. Studies on noninfectious virus occurring within cells that are producing fully infectious virus. Virology 1: ) Huang, A.S., and Baltimore, D Defective viral particles and viral disease process. Nature 226: ) Kingsbury, D.W., Portner, A., and Darlington, R.W Properties of incomplete Sendai virions and subgenomic viral RNAs. Virology 42: ,

10 784 A. MAEDA ET AL 6) Kohn, A Polykaryocytosis induced by Newcastle disease virus in monolayers of animal cells. Virology 26: ) LaMontagne, J.R., Schiller, J.G., Thacore, H.R., Feingold, D.S., and Youngner, J.S Infective and noninfective hemagglutinating particles of Newcastle disease virus: Biological and chemical characterization. J. Virol. 16: ) Lowry, O.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: ) Maeno, K., Yoshii, S., Nagata, I., and Matsumoto, T Growth of Newcastle disease virus in a HVJ carrier culture of HeLa cells. Virology 29: ) Maeno, K., and Kilbourne, E.D Developmental sequence and intracellular sites of synthesis of three structural protein antigens of influenza A2 virus. J. Virol. 5: ) Ouchterlony, O Diffusion-in-gel methods for immunological analysis. Prog. Allergy V: ) Roman, J.M., and Simon, E.H Defective interfering particles in monolayer-propagated Newcastle disease virus. Virology 69: ) Rott, R., Reda, I.M., and Schafer, W Isolation and characterization of hemagglutinating noninfectious particles produced during multiplication of Newcastle disease virus (NDV). Virology 16: ) Rott, R., and Schafer, W Cellular biology of myxovirus infections, p In Wolstenholme, G.E.W., and Knight, J. (eds) CIBA Foundation Symposium, J. & A. Churchill, Ltd., London. 15) Salk, J.E A simplified procedure for titrating hemagglutinating capacity of influenza virus and the corresponding antibody. J. Immunol. 49: Bio- Requests for reprints should be addressed to Dr. Makoto Matsumoto, Department of chemistry, Shizuoka College of Pharmacy, Oshika, Shizuoka-shi 422, Japan.