Sendai Virus) in L Cells

Japan. J. Microbiol. Vol. 18(5), 349-355, 1974

Factors Influencing the Establishment of Persistent Infection of HVJ (Sendai Virus) in L Cells

Kazuo SUGAMURA, Hideki TOZAWA1, Morio HOMMA2, and Nakao ISHIDA Departmentof Bacteriology,Tohoku UniversitySchool of Medicine,Sendai

(Received for publication, February 25, 1974)

ABSTRACT

Infection of a subline of L cells adapted to grow in suspension (Ls) with Fushimi strain of HVJ (HVJ-F) resulted in a virus carrier state. Ls cells, when cultured in monolayer, showed morphological changes following infection of HVJ-F and were detached from the glass wall. However, when the detached cells were transferred to a new environment of suspension culture within 5 days after infection, the carrier state was again established. HVJ-F caused only lethal infection in L cells maintained exclusively in monolayer (Lm). On the other hand, both Ls and Lm, irrespective of their culture conditions, were lethally infected by Nagoya 1-60 strain of HVJ. The overall results showed that culture condition as well as the kind of host cells or virus strains is an important factor regulating the establishment and maintenance of the virus carrier state.

Several paramyxoviruses and pseudopara- MATERIALS AND METHODS myxoviruses are known to induce virus car- Viruses. Two strains of HVJ, Fushimi rier states in certain cells [1, 2, 4, 8, 9, 14, 15, 18]. Cells clonally isolated from some of the (HVJ-F) and Nagoya 1-60 (HVJ-N), were carrier cultures can propagate continuously employed. HVJ-F, which has been main- with harboring viral antigens and often tained in our laboratory for nearly 20 years by allantoic passages, cannot produce plaques excrete infectious viruses [11, 15,17]. Factors influencing the establishment of the carrier in calf kidney cells. HVJ-N, which produces state seem to be the kind of virus, that of cells plaques in calf kidney cells [12], was ob- and combination of the two, since a virus tained through the courtesy of Dr. Matsu- which induces a virus carrier state in one cell moto of Nagoya University. These viruses line induces lethal infection in other cell lines were propagated in 10-day-old embryonated chicken eggs. [8, 10, 14]. The present experiments were conducted Titration of virus. Virus infectivity was to study factors which influence the establish- assayed by inoculating 10-day-old embryo- ment of virus carrier state using two lines of nated chicken eggs, 50% egg infectious doses L cells and two strains of hemagglutinating (EID50) being calculated by the formula of virus of Japan (HVJ; Sendai virus) under Reed and Muench. Hemagglutination (HA) different culture conditions. titration was carried out by Salk's pattern method. Cell culture. Two lines of L cells were Requests for reprints should be addressed to Dr. employed. One line (Ls) was kindly sup- Hideki Tozawa at the present address. 1 Present address: Department of Microbiology, plied by Dr. Tamura and Dr. Ozaki of Kyoto Tohoku University School of Dentistry, 4-1 Seiryo- University [13]. Ls can be propagated both machi, Sendai 980, Japan. in suspension and in monolayer. The other 2 Present address: Department of Bacteriology, line (Lm) has been passaged exclusively in Yamagata University School of Medicine, Yamagata monolayer, since it cannot grow in suspension. 990-23, Japan.

349 350 K. SUGAMURA, H. TOZAWA, M. HOMMA AND N. ISHIDA

Suspension cultures of Ls were performed by stirring in 100 ml of medium in a 200-ml flask at 37 C and the cell number was kept

between 3•~105 and 106 cells/ml by daily replenishments with fresh medium. For monolayer culture, both Ls and Lm were sown in Roux bottles. Media. The medium for suspension culture consisted of 0.5% lactalbumin hydrolysate and 0.1% yeast extract in Earle's balanced

salt solution (YLE) supplemented with 5% heated horse serum. The medium for monolayer culture consisted of YLE supplemented with 10% unheated bovine serum. After infection, cultures in both conditions were maintained in YLE containing 5% heat- inactivated horse serum. Cell count. Viable cells were counted in a

Burker-Turk hemocytometer after staining with a 0.5% trypan blue solution. Immunofluorescent staining. Anti-HVJ sera were prepared in rabbits. They were in- Fig. 1. Growth of Ls cells after infection with jected intravenously with 10 doses of 104 HA HVJ-F. Cell culture was performed in suspension units of virus at intervals of 3 days and bled as described in Materials and Methods. Ls was 10 days after the last injection. Immuno- infected with HVJ-F at an moi of 100 EID50 per

globulins were extracted from the antiserum cell. The cell number was kept between 3•¬105 by salting-out with ammonium sulfate, and and 106 cells/ml. Counting of viable cells was made after staining with trypan blue with the labeled with fluorescein isothiocyanate ac- uninfected(•›-•›) and infected cultures (•œ-•œ). cording to a modification of Marshall's method [5]. For immunofluorescent staining, cells on a cover slip were fixed with CCl4 for 30 min at 4 C and incubated with the labeled antibodies for 30 min at 37 C.

RESULTS

Characterization of Persistent Infection of Ls by HVJ-F A suspension culture of Ls (5•~105 cells/ml) was infected with HVJ-F at a multiplicity of infection (moi) of 100 EID50 per cell. The infected cells continued to grow with an ap- proximate doubling time of 24 hr for over 30 days and the growth rate was comparable to Fig. 2. Immunofluorescent staining of Ls/HVJ-F that of the control uninfected culture (Fig. 1). carrier cells. The carrier cells on the 8th day Immunofluorescent staining was made on after infection at an moi of 100 EID50 per cell were the 5th and 20th days after infection and stained as described in Materials and Methods. in each instance a total of 10000 cells from 10 preparations were observed. As illus- when compared with those obtained in the trated in Fig. 2, all cells were infected and had usual lytic infection system (Fig. 3). The viral antigen in the cytoplasm. Titers of both results also revealed that the virus tended to the cell-associated and the released virus in accumulate in cells by 48 hr after infection. culture fluid were very low at any given time An immediate release occurred at 96 hr and PERSISTENT INFECTION OF L CELLS WITH HVJ 351 thereafter a balance seemed to be held be- result showed that all cells had viral antigen tween the virus production and release. in the cytoplasm, indicating that the anti- When cells from the culture on the 8th day viral serum was not effective to cure the virus of infection were inoculated into embryo- carrier state. Moreover, interferon produc- nated chicken eggs, four vital cells roughly tion did not seem to be involved in mainte- corresponded to one infective center. How- nance of the virus carrier state, since vesicular ever, the infectivity of the cells was readily stomatitis virus (VSV) production was not abolished by freezing and thawing (Table 1). affected in the carrier culture (Table 2). To see the effect of antiviral serum on main- The overall observations showed that Ls tenance of the virus carrier state, an Ls was infected persistently by HVJ-F in the culture infected with HVJ-F for 10 days was condition of suspended culture. The carrier incubated for additional 7 days in the pre- system thus established was designated as sence of 2% anti-HVJ rabbit serum with an Ls/HVJ-F. HI titer of 104/ml and the cells were sub- jected to fluorescent antibody staining. The Conditions Leading L Cells to Carrier Culture It was evident from the foregoing results that HVJ-F did not cause lysis of Ls in suspension. This exhibited a striking con- trast to our earlier observations that infection of L cells in monolayer with HVJ led to cell lysis [6]. Two explanations might be pos-

Table 1. Infectivity of Ls/HVJ-F carrier cellsa)

Fig. 3. Time course of virus production in the

Ls/HVJ-F carrier culture at an early stage of in- a) Ls/HVJ-F carrier cells on the 8th day fection. An Ls suspension culture was infected

with HVJ-F at an moi of 100 EID50 per cell for 1 hr after infection were washed carefully by a low at 37 C and then washed with medium con- speed centrifugation, suspended in medium so taining anti-HVJ rabbit serum to remove residual as to contain the indicated cell number and virus. Viruses produced were titrated for EID50 inoculated into 10-day-old embryonated

and HA, and the quantities in 1 ml of fluid con- chicken eggs. One-half of the cells was sub- taining 5•~105 cells calculated. EID50 (•›-•›) jected to three cycles of freezing and thawing and HA (•›---•›) after 3 cycles of freezing and before inoculation. b) The numerator indicates the number of thawing of the whole culture. EID50 (•œ-•œ) and HA (•œ---•œ) after removal of the cells by eggs infected, and the denominator the num- centrifugation. ber of eggs tested.

Table 2. Production of VSV in the Ls/HVJ-F carrier culturea)

a) The carrier culture 12 days after infection, and uninfected Ls cells as a control, were infected with Indiana strain of vesicular stomatitis virus (VSV) at an moi of 2 PFU per cell. After incubation for 24 hr, serial tenfold dilutions of the culture fluids were titrated in tube cultures of Lm cells, using 6 tubes per dilution. b) The numerator indicates the number of tubes showing cytopathic effect (CPE) and the denominator the number of tubes tested. 352 K. SUGAMURA, H. TOZAWA, M. HOMMA AND N. ISHIDA sible for the above apparent conflict. The tion, the release of HA into culture fluid first one is that Ls has different properties remained at a low level (23 HAU/ml) with from other lines of L cells. This is likely to be Ls, while it reached a high level (27 HAU/ml) the case because Ls is known to have under- with Lm (Table 3). gone a selection during the course of its The above results not only clearly demon- establishment [13]. The alternative one is strated differences between Ls and Lm in that L cells in suspension behave differently reactivity to HVJ-F but also implied that from those in monolayer upon infection with the morphological changes of L cells due to HVJ. Because of the fact that Ls can grow HVJ infection do not necessarily lead the in monolayer as well as in suspension, the cells to lysis as judged by the trypan blue latter possibility could easily be tested by the dye exclusion test. following experiment. Transfer of Culture Conditionsof the InfectedCells Infection of Ls in Monolayer Condition To see whether infected Ls cells in mono- A monolayer culture of Ls was infected layer which exhibited morphological changes with HVJ-F at an moi of 100 EID50 per cell. were actually able to divide, the following In parallel with this experiment, a mono- experiment was conducted. Ls in monolayer culture of Lm was also infected and layer was infected with HVJ-F at an moi of served as a control. After removal of re- 100 EID50 per cell, and cells detached from sidual unadsorbed virus, cytological changes the glass wall were collected at various time and development of viral antigen were fol- intervals by gentle shaking. The cells were lowed during the subsequent incubation. immediately transferred to a new environ- At 24 hr after infection, all cells in both ment in an attempt to grow them in suspen- cultures were rounded up and began to sion. The cells transferred within 5 days slough off the glass wall. Immunofluores- after infection started to increase in number cent staining demonstrated that all cells were without an appreciable lag period and producing viral antigen in the cytoplasm but continued to propagate at the same rate as no significant differences were revealed in the cells in uninfected Ls culture. Immuno- the fluorescent features between the cells of fluorescent staining again detected viral the two cultures. Striking differences were antigen in the cytoplasm of all cells and HA found, however, between Ls and Lm when titer of virus released into culture fluid also the dye exclusion test was done by trypan remained on a low level (23 HAU/ml). blue staining. Apparently none of the cells However, unless the infected cells were from Ls were stained by the dye at least transferred to the new environment, all of within 5 days after infection while most of the cells were eventually detached from the the cells from Lm were stainable. In addi- glass wall and finally became stainable by trypan blue. An attempt to recover viable Table 3. Infection of Ls and Lm cells with cells from Lm cells infected in monolayer by HVJ-F transfer to suspension culture was unsuccess- ful. The above results showed that rounding of cells in the Ls monolayer culture infected with HVJ-F was essentially different from that observed in Lm culture; the cells lost the ability of attaching to the glass wall, but could continue to grow in a suitable circum- stance.

a) The viruses released into culture fluid Lethal Response of L Cells at Infection with were measured at 72 hr after infection. The HVJ-N titer was expressed by that of a culture which contained 106 cells/ml. The results hitherto described raised a b) Cells were observed 5 days after infec - question of whether Ls cells were to bring tion. about a carrier state upon infection with PERSISTENT INFECTION OF L CELLS WITH HVJ 353

Table 4. Infection of Ls and Lm cells with HVJ-N

a) The viruses released into culture fluid were measured at 72 hr after infection. The titer was expressed as in Table 3. b) Cells were observed 5 days after infection.

titers of virus released into culture fluid were all as high as 27 HAU/ml at 72 hr after infec-

tion (Table 4). These results indicated that infection with HVJ-N eventually led both Ls and Lm cells to death irrespective of their culture conditions.

Fig. 4. Growth of Ls cells infected with HVJ-N. DISCUSSION An Ls suspension culture was infected with HVJ-N Infection of Ls cultured in suspension with at an moi of 100 EID50 per cell. Cell culture and counting of viable cells were as described in Fig. 1. HVJ-F did not cause cell death but resulted

Uninfected cells (•›-•›) and infected cells in a virus carrier state. This carrier culture (•œ-•œ). should be placed in the same category as the regulated infection defined by Walker [16] every strain of HVJ. In the following ex- on the basis of the following criteria: 1) the periment, HVJ-N, which was considered to cells grew continuously with harboring viral be more virulent than HVJ-F on the basis of antigen. 2) Neither antiviral serum nor plaque-forming ability in calf kidney cell interferon was required for establishment or culture [12], was substituted for HVJ-F. for maintenance of the virus carrier state. 3) Cultures of Ls both in monolayer and in The presence of the antiviral serum in suspension and those of Lm in monolayer culture fluid could not yield cells free of viral were infected with HVJ-N at an moi of 100 antigen. EID50 per cell as described, and cytological Infection with HVJ-F caused morpholog- changes and development of viral antigen ical changes of the cells in Ls which had been were pursued. cultured exclusively in monolayer, the feature In the case of Ls in suspension, the cells being very similar to that observed in lethally continued to increase in number for 3 days infected L cells. However, when such cells after infection at the same rate as uninfected were transferred to the condition of sus- control cells. This was, however, followed pended culture, they were able to grow by an abrupt decrease on the 4th day and no continuously at the same growth rate as viable cells could be detected on the 7th day control uninfected cells. In a biological sense, (Fig. 4). When cultures of Ls and Lm in one of the major differences from the unin- monolayer were infected with the same virus, fected cells was that the cells lost the ability rounding of the cells appeared within 24 hr to adhere to the glass wall. This phenome- and most of the cells in both cultures became non may have similarity to the concept, stainable by trypan blue on the 5th day. •g paralysis,•h proposed by Watkins [19, 20]: Immunofluorescent staining detected viral HeLa cells infected with herpes simplex antigen in all cells from both cultures and HA virus lost the ability to spread on the glass 354 K. SUGAMURA, H. TOZAWA, M. HOMMA AND N. ISHIDA wall. These phenomena may be due to a tions, it may not be concluded that an modification of cell membrane by virtue of immediate release of virus produced from the virus infection through some unknown mech- cell is responsible for induction of a carrier anism. state. 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