Comparative Studies of Rabies and Sindbis Virus ... - CiteSeerX

J.

gen. Virol. (1984), 65, 1645-1653. Printedin Great Britain

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Key words: rabies virus/Sindbis virus~human neuroblastoma cell/IFN

Comparative Studies of Rabies and Sindbis Virus Replication in Human Neuroblastoma (SYM-I) Cells that Can Produce Interferon By Y O S H I K A Z U

H O N D A , A K I H I K O K A W A I * AND S E I I C H I MATSUMOTOt Institute for Virus Research, Kyoto University, Kyoto 606, Japan (Accepted 4 June 1984)

SUMMARY K-104 cells, a cloned cell line derived from a human neuroblastoma (SYM-I), were induced by rabies HEP-Flury virus to release large amounts of interferon, and the resulting antiviral activity significantly suppressed the rabies virus replication. The role of endogenous interferon was confirmed by treatment with anti-interferon antibody which increased the yield of progeny virus. The virus yield in the second undiluted passage through K-104 cells was much less than that in the first passage, because of the antiviral state initiated by brief contact of interferon present in the virus inoculum with cells during the short period of virus adsorption. When the m.o.i, was relatively low, as in the third undiluted passage, the effect of interferon present in the inoculum was enhanced and most of the infected cells survived but were shown to be in a state of persistent infection. Defective interfering (DI) particles did not accumulate rapidly during these three undiluted passages. When Sindbis virus was used for infection, the endogenous interferon system of K-104 cells was not activated during 12 undiluted passages. However, on the 12th passage, the yield began to decline due to the generation and accumulation of DI particles.

INTRODUCTION Rabies virus introduced through bite wounds made by rabid animals into extra-neural tissues is known to pass via a peripheral nerve route to the central nervous system, eventually producing fatal encephalitis (Murphy, 1977). A long and variable incubation period is a characteristic feature of rabies virus infection. The outcome of infection is determined during this incubation period, i.e. the time before the virus spreads to the central nervous system, since the virus is believed to stay at or near the site of its introduction for most of the incubation period (Baer & Cleary, 1972). Many factors are involved in determining the result of virus infection, for example the virulence and dose of the virus, the density of susceptible cells at the site of introduction, the presence of locally produced interferon induced by virus infection and other specific and nonspecific defence mechanisms of the host. Interferon is thought to be one of the most important of these factors because it starts to work in the early phase of infection. Rabies virus has been shown to be sensitive to the antiviral activity of exogenous interferon (DePoux, 1965; Stewart & Sulkin, 1968; Karakuyumchan & Bektemirova, 1968) and to induce interferon production in cultured cells (Yoshino et al., 1966; W iktor et al., 1972 a) and in animals (Stewart & Sulkin, 1966; Wiktor et al., 1972b, 1976; Baer et al., 1977; Mifune et al., 1980). Local treatment of bite wounds on one leg with interferon or with interferon inducers has been shown to be more effective in preventing the development of rabies than treatment with interferon or its inducers at other sites (Postic & Fenje, 1971 ; Fenje & Postic, 1971 ; Harmon & Janis, 1975). Studies of the efficacy of rabies vaccine for post-exposure prophylaxis have revealed that the potency of the vaccine largely depends on its ability to induce interferon production (Wiktor et al., 1976; Baer & Yager, 1977). Furthermore, Baer et al. (1977, 1979) t Died March 13, 1983. 0022-1317/84/0000-6039 $02.00 © 1984 SGM

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Y. HONDA, A. KAWAI AND S. MATSUMOTO

showed that post-exposure treatment of mice and rhesus monkeys with a combination of vaccine and interferon, or an interferon inducer, was more effective than treatment with vaccine alone. If interferon is produced in animals in the early phase of infection, it will suppress virus replication in extra-neural tissues, which may result in limiting of virus spread into neural tissues (Stewart, 1981). To ascertain this, more detailed analyses on the effect of interferon on virushost cell interactions in vitro as well as in vivo are necessary. Rabies virus infection in cultured cells that have fully functional interferon systems should provide a useful model for investigating the effect of interferon on the interaction of rabies virus and host cells in nature. No detailed studies of such a model system in vitro, however, have been reported. One reason may be the lack of adequate host cells that are sensitive to rabies virus and have the ability to produce interferon. We recently found that human neuroblastoma SYM-I (clone K-104) cells are sensitive to rabies virus infection and can produce much interferon. Thus, studies of rabies virus replication in these cells offer a good model from which the role of the interferon system in vivo could be deduced. We report here the effect of interferon on rabies virus infection in K-104 cells and describe the interference phenomena observed during undiluted viral passages through these cultures in comparison with passages of Sindbis virus (another enveloped R N A virus) which replicates rapidly in K-104 cells and is known to be highly sensitive to interferon.

METHODS Viruses. Virus stocks of the cloned HEP-Flury strain of fixed rabies virus were prepared by a one-cycle amplification of a mixture of plaque isolates in BHK-21 cell culture. The content of defective interfering (DI) particles was determined by the interference focus assay (Kawai & Matsumoto, 1982). Plaque-cloned vesicular stomatitis virus (VSV), New Jersey serotype, was propagated in monolayer cultures of BHK cells (Kawai, 1977). Sindbis virus was obtained from Dr Y. Kawade (Fukada et aL, 1968) and was propagated in BHK cells by diluted passages. The content of DI particles in Sindbis virus preparations produced by serial undiluted passages was determined by the extent of interference with normal virus replication as described by Schlesinger et al. (1972). The Miyadera strain of Newcastle disease virus (NDV) was grown in the allantoic cavities of 11-day-old embryos, whose allantoic fluids were harvested 2 days after inoculation. Cell cultures and media. A human neuroblastoma cell line, SYM-I, established by Dr M. Sekiguchi (Institute of Medical Science, the University of Tokyo, Tokyo, Japan) was obtained from Dr K. Hayashi (Institute of Medical Science, the University of Tokyo) and cloned in our laboratory. Clone K-104 was used throughout this research because its fiat morphology was favourable for microscopic observations after histological staining. K-104 cells were propagated in modified Eagle's MEM containing twice the normal concentrations of amino acids and vitamins and was supplemented with 10% foetal calf serum (FCS). HeLa cells were cultivated in Eagle's MEM containing 5% bovine serum (BS) and BHK-21 cells in Eagle's MEM supplemented with 5 ~ BS and 10~ tryptose phosphate broth (Difco). Infectivity assay. The infectivity of rabies virus was determined by plaque formation assay with agarosesuspended BHK-21 cells (Sedwick & Wiktor, 1967). VSV and Sindbis virus were titrated by the ordinary plaque formation method on BHK cells. Interferon. NDV-induced human beta-type interferon (HuIFN-fl) from K-104 cells was used after a 4-day exposure to acid (pH 2.0) followed by clarification by ultracentrifugation at 70000 to 100000g for 60 min, but without further purification. Interferon assay. HulFN titres were determined by a plaque reduction method using HeLa cells and VSV as the challenge virus. The titres were expressed in International Units (IU) by calculating the reciprocals of the dilution that showed 50 % reduction of plaque formation and comparing them with a standard interferon preparation (N IH G-023-902-527). The rabies viruses contained in the interferon assay samples which had been clarified beforehand by ultracentrifugation (as described above) were inactivated by u.v. light or by neutralization with rabbit antiserum to rabies virus. The reference standards for interferon were supplied by Drs Y. Yamamoto and Y. Watanabe of this Institute. Antisera. Anti-rabies serum was prepared by immunizing rabbits with purified virions of the HEP-Flury strain (Kawai & Matsumoto, 1982). The highly specific, anti-HulFN-fl globulin was received from Dr Y. Kawade of this Institute; it was originally prepared by Dr B. Dalton (The Medical College of Pennsylvania, Philadelphia, Pa., U.S.A.) from sera of sheep immunized with purified poly(I).poly(C)-induced human fibroblast interferon. Its neutralization titre, defined as the reciprocal of the sample dilution that reduces 16 Laboratory Units (LU) of interferon per ml to 1 LU/ml (Kawade, 1980), was 3000.

Rabies virus replication and interferon

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RESULTS

Effects of serial undiluted passages on the yield of infeetious virus in human neuroblastoma K-104 cells When the HEP-Flury strain of fixed rabies virus was passed serially through the K-104 cell culture, a sharp reduction in virus yields was observed (Fig. 1). The original stock virus was prepared by a single cycle of replication of plaque isolates in BHK cell culture, and was almost free from DI particles as determined by the interference focus formation assay. At the first passage, the stock virus was inoculated onto monolayers of K-104 cells at an m.o.i, of 2 p.f.u./cell. Virus yield was somewhat variable, and 106 to 107 p.f.u./ml of progeny virus was usually obtained by 50 to 60 h after infection, but this titre was much less than the virus yield from BHK cells infected with the same virus. In the second passage of undiluted virus infection, in which the m.o.i, was about 2 p.f.u./cell, the virus yield was only about 1/10 that of the first passage. In the third undiluted passage, in which the input dose of virus was inevitably decreased to as little as 0.2 p.f.u./cell, the progeny virus yield was only about 1/10000 that of the first passage. Through the second passage, severe c.p.e, was caused by rabies virus infection in K-104 cell culture, but at the third passage almost all c.p.e, was suppressed. Infection in this culture, however, resulted in the establishment of a persistent infection which could be maintained successfully by serial subculture at intervals of 3 to 4 days, and the titre of the infectivity produced was usually as low as a thousandth or less of that obtained at the first virus passage.

Nature of the interfering factor in rabies virus-infected K-104 cell cultures The decrease in infectious virus yields during serial virus passages in K-104 cells seemed, however, too rapid to be explained only by the production of DI particles. In fact, the titre of the DI particles was not high, as shown by the interference focus formation assay (data not shown), suggesting that this interference phenomenon was mediated by factors other than DI particles. As shown in Fig. 2, the interfering activity was still observed in the supernatant when the sedimentable fraction was removed by ultracentrifugation at 77000g for 60 min. When the inoculum was prepared by resuspending the virus pellet in the supernatant of the same centrifuge tube, the interfering activity was restored to the original level. In contrast, when these were inoculated onto BHK cell monolayers, no suppression of progeny virus production took place (data not shown). Further investigation to characterize this interfering factor showed that its properties were completely consistent with those of interferon : the interfering factor (i) was not sedimented by ultracentrifugation for 120 min at 100000 g, (ii) was stable for 2 days at a low pH, (iii) was active on HeLa cells (another type of human cell) but not on hamster BHK cells, (iv) was active in inhibiting the replication of VSV and Sindbis, (v) was inactivated by treatment with trypsin, and (vi) was neutralized by specific antibodies prepared against HuIFN-/~. Time courses of the production of infectious virus and interferon in rabies virus-infected K-104 cell cultures in the first and second viral passages showed that, in both cases, interferon became detectable in the culture medium at the 12th h of infection and continued to accumulate until the 60th to 70th h when virus growth ceased and c.p.e, appeared, reaching a maximum titre of 2 x 103 to 3 x 103 IU/ml.

Undiluted passages of Sindbis virus in K-104 cell culture Sindbis virus was chosen because it replicates well in K-104 cells and is highly sensitive to the antiviral activity of interferon exerted in this cell line. Results of serial undiluted passages showed no decrease in the yield of infectivity until the llth passage (Fig. 1). In fact, no interferon was detected in the culture medium from any virus passage, and the decrease in virus titre observed in the 12th passage was due to interference caused by DI particles which gradually accumulated up to the 1lth passage (data not shown).

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Y. H O N D A , A. K A W A I AND S. MATSUMOTO

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Fig. 1. Undiluted passages of rabies virus and Sindbis virus through cell cultures. Except in the first passages, monolayers of K-104 or BHK cells which had been placed in 3.5 cm plastic Petri dishes were inoculated with the undiluted culture fluid from each preceding virus passage after the first passage. Rabies virus stock prepared by a one-cycle amplification of plaque isolates in BHK cells was used at an m.o.i, of 2 p.f.u./cell for the inoculum in the first virus passage. Undiluted culture fluid from BHK cells infected with diluted Sindbis virus stock was used as the inoculum for the first passage. After virus adsorption for 60 rain at room temperature, cells were washed with PBS then incubated at 36 °C in Eagle's MEM plus 2% FCS. Their culture fluids were harvested at 12 h intervals up to 72 h (rabies virus) or at 12 h (Sindbis virus) of infection. Infectivity was determined by plaque formation following undiluted passages of Sindbis virus through K- 104 cells (O), and of rabies virus through BHK cells (IS]) or K-104 cells (A). Fig. 2. Demonstration of a soluble interfering factor in culture fluids from rabies virus-infected K-104 cells. Culture medium harvested at the 50th h of infection of the first virus passage was divided into four parts; three were ultracentrifuged at 77000 g for 60 min, then one virus pellet was resuspended in the supernatant of the same culture medium, another was resuspended in PBS containing 2 % FCS, and the third was resuspended in PBS containing 2% FCS and 200 IU HulFN-fl; the fourth received no treatment. Each preparation was inoculated onto confluent K-104 cell monolayers on plastic plates. As a control, a pair of plates of K-104 cells were inoculated with original virus stock grown in BHK cells. The multiplicity of each infection was adjusted to 2 p.f.u./cell. After 1 h stirring on a rocker platform at room temperature for virus adsorption, cells were washed three times with warm PBS then incubated at 36 °C in Eagle's MEM plus 2% FCS. Culture fluids were harvested at the intervals specified then assayed for infectivity: O, plaque-cloned HEP-Flury virus amplified once in BHK cells; t-q, the culture medium of the first virus passage through K-104 cells; m, a virus suspension prepared by sedimenting viruses by ultracentrifugation in the culture fluid of the first virus passage then resuspending them in the supernatant from the same centrifuge tube; O, a virus suspension prepared by sedimenting viruses as in (m) but resuspending them in PBS containing 2% FCS; A, a virus suspension prepared by sedimenting viruses as in (11) but resuspending them in PBS containing 2 ~ FCS and 200 IU HulFN-fl.


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Fig. 3. Kinetics of the development of an antiviral state shown by resistance to Sindbis virus superinfection of K-104 cells after rabies virus infection or HuIFN-fl treatment. Monolayers of K- 104 cells in 3.5 cm dishes were inoculated with HEP-Flury rabies virus (m.o.i. 2 p.f.u./cell) or HulFN-fl (200 IU/dish) then incubated for 1 h at room temperature. The infected cells were washed three times with warm PBS then incubated at 36 °C in Eagle's MEM containing 2 ~ FCS. A sample of the dishes was challenged with Sindbis virus at an m.o.i, of 2 p.f.u./cell at the indicated intervals. Sixteen h after the challenge, culture fluids were harvested for the Sindbis virus assay by plaque formation and, at the same time, the number of cells in each culture was determined. The same experiment was performed on BHK cells omitting interferon treatment. II, Rabies virus-infected K-104 cells; I--1, uninfected, untreated control K-104 cells; A, interferon-treated K-104 cells; 0 , rabies virus-infected BHK cells; O, uninfected, untreated control BHK cells. Fig. 4. Effect of anti-HulFN-fl globulin on rabies virus growth in K-104 cells. K-104 cells which had been infected with rabies virus at an m.o.i, of 4 p.f.u./cell were incubated with, or without, sheep antiHulFN-fl globulin. This was added to the medium at a final concentration of 150 units/ml (see Methods) 1 h after infection. Culture fluids were harvested at the intervals specified then assayed for infectivity. O, Untreated control culture; O, antibody-treated culture.

Kinetics of the inhibitory action of interferon at the first passage of rabies virus in K-104 cells B a s e d o n t h e a b o v e o b s e r v a t i o n s a n d o t h e r e v i d e n c e t h a t r a b i e s v i r u s usually t a k e s 50 to 60 h for o n e cycle o f r e p l i c a t i o n , e v e n in s u c h h i g h l y s u s c e p t i b l e cells as B H K - 2 1 w h i c h l a c k t h e a b i l i t y to p r o d u c e i n t e r f e r o n ( K a w a i & M a t s u m o t o , 1977), we s p e c u l a t e d t h a t t h e e n d o g e n o u s i n t e r f e r o n i n d u c e d in K - 1 0 4 cell c u l t u r e b y r a b i e s v i r u s i n f e c t i o n m i g h t s u p p r e s s t h e v i r u s r e p l i c a t i o n in p r o g r e s s i n t h e s a m e culture. T o a s c e r t a i n w h e t h e r t h i s w e r e so, we i n v e s t i g a t e d t h e k i n e t i c s o f t h e d e v e l o p m e n t o f a n a n t i v i r a l s t a t e i n r a b i e s v i r u s - i n f e c t e d cells. T h e d e g r e e o f a n t i v i r a l a c t i v i t y w a s m o n i t o r e d b y t h e cell's r e s p o n s e to a c h a l l e n g e b y S i n d b i s v i r u s s u p e r i n f e c t i o n . Fig. 3 d e p i c t s t h e t i m e c o u r s e s o f c h a n g e s in t h e s u s c e p t i b i l i t y o f K - 1 0 4 cells to s u p e r i n f e c t i o n w i t h S i n d b i s v i r u s a f t e r r a b i e s

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Y. HONDA, A. KAWAI AND S. MATSUMOTO I

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Fig. 5. Effect of anti-HulFN-fl globulin on the development of resistance to Sindbis virus superinfection of rabies virus-infected K-104 cells. Monolayers of K-104 cells infected with, or without, rabies virus (m.o.i. 4 p.f.u./cell) were incubated with, or without, anti-HulFN-fl globulin (final concentration 150 units/ml). Other conditions of Sindbis virus superinfection are as given in Fig. 3. 0 , Rabies virus-infected culture; &, rabies virus-infected and anti-IFN-treated culture; A uninfected but antiIFN-treated culture; O, uninfected, untreated control culture.

virus infection or interferon treatment. Resistance to Sindbis virus superinfection became a p p a r e n t about 18 h after rabies virus infection. In interferon-treated K-104 cells, it took 6 h to develop an antiviral state. In contrast, rabies virus-infected B H K cells, which do not produce interferon, did not develop resistance to superinfection by Sindbis virus. These results suggest that rabies virus does not interfere directly with the replication of superinfecting Sindbis virus and that rabies virus-infected K-104 cells might gradually enter the antiviral state after the lapse of two periods, i.e. the interval needed for inducing interferon production (12 h) and that for the development of an antiviral state (6 h), 18 h in total, which is coincident with the time o f manifestation of resistance of K-104 cells to Sindbis virus superinfection (Fig. 3). This speculation was confirmed by introducing specific anti-interferon antibody into the rabies virus-infected K-104 cell culture. W h e n 300 units of a n t i - H u l F N - f l (see Methods) was added to an infected K-104 cell culture, a constant and greatly enhanced production of infectious virus was found (Fig. 4), although the yield of infectivity remained tenfold lower than that from B H K cells infected with the same virus. In addition, the development of resistance to Sindbis virus superinfection in rabies virus-infected K-104 cells was almost completely suppressed in this culture (Fig. 5).

The role of interferon in the second and third undiluted passages When K-104 cells were infected with an inoculum prepared by resuspending a virus pellet in phosphate-buffered saline (PBS) that contained 100 I U / m l of interferon, the virus yield was reduced to l / I 0 of that of the control infection which had no exogenous interferon (Fig. 2). This clearly demonstrates that brief treatment with a small amount of interferon during the short


1651

period of virus adsorption is enough to initiate the development of an antiviral state. In addition, the endogenous interferon produced after infection with rabies virus may also act together with the interferon present in the inoculum to enhance the development of the antiviral state during the second virus passage. In the third undiluted passage, at least three elements must function: the interferon present in the inoculum, the endogenously produced interferon after rabies virus infection and the inevitably low m.o.i, due to the low virus yield in the second undiluted passage. Infection at a low m.o.i, would give the host cell enough time to establish a strong antiviral state; thus, the virus yield from the third undiluted virus passage would be greatly reduced as shown in Fig. 1. DISCUSSION

The endogenous interferon induced by rabies virus infection was shown to interfere markedly with virus replication in progress in the same culture. These consequences may be explained by properties of the virus-host cell system: (i) it takes a very long time for rabies virus to replicate (50 to 60 h for one cycle of multiplication), (ii) rabies virus causes a gradual reduction in the host cell's macromolecule synthesis (Matsumoto, 1974; Madore & England, 1975) that would allow the cell to produce a high titre of interferon, (iii) rabies virus is very sensitive to the antiviral activity of interferon. Thus, rabies virus-infected K-104 cells might gradually enter the antiviral state before the beginning of vigorous virus production. Similar findings of the role of endogenous interferon in some other virus-host cell systems have been reported in which the application of anti-interferon antibody treatment was included (Vil~ek et al., 1977; Nagai et al., 1981; Jacobson & McFarland, 1982; Linnavuori & Hovi, 1983). Serial undiluted passage of rabies virus through BHK cell cultures usually results in the generation and accumulation of DI particles which interfere with the production of the progeny virus in subsequent virus passages, but the decrease in virus titre was not as rapid as that seen in K-104 cell cultures and the extent of the decrease was limited to about 10~ to 105 p.f.u./ml (Kawai et al., 1975; Kawai & Matsumoto, 1977). In contrast, DI particles did not accumulate rapidly during three undiluted passages through K-104 cells although virus yields were greatly reduced. The replicative cycle of Sindbis virus is very short compared to that of rabies virus, and causes a strong shut-off of macromolecule synthesis (Strauss et al., 1969). This may account for the lack of participation of endogenous interferon in Sindbis virus-infected K-104 cells and, as a result, an extremely high yield of progeny virus was obtained. The changes in the yield of infectivity during undiluted virus passage through K-104 ceils was due to the generation and accumulation of DI particles as seen in another host cell system (Schlesinger et al., 1972). Sindbis virus has been reported, however, to induce interferon production in certain host cells (Marcus & Fuller, 1979). One of the causes of the difference in cell response might be the extent of host cell shut-off. The fact that the endogenous interferon produced following rabies virus infection in turn affects subsequent virus multiplication leads us to assume that similar consequences might take place during infection in vivo, especially in the period of extra-neural infection. The investigation of local interferon production in extra-neural tissues is very important because the infecting virus is believed to stay at, or near, the site of its introduction for most of its long incubation period (Baer & Cleary, 1972). However, interferon production by rabies virus in animals has been so far demonstrated only in infected brains of hamsters and mice (Stewart & Sulkin, 1966; Wiktor et al., 1972a). The interferon system of K-104 cells could not eliminate the infecting virus, and it initiated the establishment of persistent infection as noted in a report on VSV persistent infection (Sekellick & Marcus, 1980). The properties of a persistent rabies virus infection in K-104 cells will be described elsewhere. Our results with K-104 cells may not be directly applicable to rabies virus infection in animals, because even if viruses survive in the form of a persistent infection, most of the persistently infected cells might be eliminated by the host's other defence mechanisms. However, the fact that rabies virus could persist in cells with the aid of endogenous interferon must not be ignored when considering the post-exposure prophylaxis of rabies by treatment with interferon, or its inducers, alone.

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Y. H O N D A , A. K A W A I A N D S. M A T S U M O T O

We thank Drs Y. Kawade, Y. Y a m a m o t o and Y. Watanabe for their generous gifts of antibody prepared against h u m a n interferon, reference standards of h u m a n interferon and a Sindbis virus stock, Ms M. K u m a m o t o and T. Sugimoto for their techniqal assistance, Ms P. Y a m a d a and Mr D. Mrozek for their help in revising the manuscript and Ms M. Tsutsumi for typing the manuscript. This work was supported in part by Grants-in-Aid for scientific Research B (no. 56480136) and for Co-operative Research A (no. 56370013; Dr H. Shibuta, director) from the Ministry of Education, Science and Culture, Japan.

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(Received 5 January 1984)