Temperature-Sensitive Mutants Isolated from Hamster and

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1 JOURNAL OF VIROLOGY, Nov. 1975, p Copyright i 1975 American Society for Microbiology Vol. 16, No. 5 Printed in U.S.A. Temperature-Sensitive Mutants Isolated from Hamster and Canine Cell Lines Persistently Infected with Newcastle Disease Virus JULIUS S. YOUNGNER* AND DIANE 0. QUAGLIANA Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania Received for publication 7 July 1975 Evidence is presented which confirms that temperature-sensitive (ts) mutants with an RNA- phenotype are spontaneously selected in persistent infection of cell lines with Newcastle disease virus. Persistently infected BHK-21 cells, maintained since 1973, produce no interferon and are completely susceptible to vesicular stomatitis virus. Persistent infection of a canine kidney cell line (MDCK) terminated with destruction of all cells at about 100 days. Even under these conditions, a high proportion (33%) of RNA- temperature-sensitive mutants was present in the virus population 60 days after the infection was initiated. Previous results from this laboratory have ture bottles occurred 38 days after the infection shown that temperature-sensitive (ts) mutants was initiated. Confluent cell monolayers were were spontaneously selected in L cells persistently infected with several strains of Newcastle and subsequent passages were made from one generally formed 4 to 7 days after subculture disease virus (NDV) (3, 4). Evidence suggested culture bottle to 5 or 10 new bottles. After that the emergence of ts mutants in persistent several months, scraping the cells from the glass infection is not random and that these mutants with a rubber policeman was utilized for cell play a role in the establishment and maintenance of the viral carrier state (4, 5). This report The infected cell lines periodically formed passages instead of trypsin. shows that the Herts strain of Newcastle disease giant cells and syncytia, often resulting in a virus can establish persistent infection in cell crisis with significant loss of cell viability. In lines from species other than mice and that in such cases, the cultures were fed frequently with each case ts mutants are spontaneously selected fresh medium until the cells multiplied sufficiently for a cell passage to be made. There was in the carrier cells. Baby hamster kidney (BHK-21) and canine no regularity in the intervals between crises. kidney (MDCK) cell lines were propagated in When the BHKNDV line had stabilized 3 Eagle minimal essential medium containing 4% months after the infection was initiated, cell calf serum. Assays for infectivity, hemagglutinin, and virus-specific RNA synthesis were presence of virus. Infective virus in the culture cultures were tested at frequent intervals for the carried out as previously described (4, 7, 8). fluid ranged from as few as 2 x 102 to as many A persistently infected line of BHK-21 cells as 1.3 x 106 PFU/ml; hemagglutinin units per (BHKNDV) was initiated in the following manner. In April 1973, cell monolayers were infected PFU/hemagglutinin unit ratios usually were ml of culture fluid ranged from 2 to 32. The with egg-grown Herts NDV (NDVO), using input from 5 x 101 to 8 x 104, compared to a ratio of multiplicities of infection (MOI) ranging from 5.6 x 105 for NDVo grown in MDCK cells. These 0.1 to Within 24 h nearly the entire results indicated that significant numbers of monolayer showed virus-induced cytopathic effects at all multiplicities. Some cells remained present in fluid from BHKNDV cells, a finding noninfective hemagglutinating particles were viable and these slowly increased in number. similar to what has been noted in persistently After many medium changes in the succeeding infected L cells (0. T. Preble, unpublished weeks, enough cells were present in cultures data). The amount of virus present could not be originally infected at an MOI of 0.1 to be correlated with the periodic crises which occurred. The number of BHKNDV cells plated for subcultured and maintained in 2-ounce prescription bottles. The first passage of cells by each plaque-forming cell was determined by a trypsinization and subdividing to several cul- method described elsewhere (7). From one to 1332

2 VOL. 16, 1975 eight cells per plaque-forming cell was the value obtained most frequently; from time to time as many as 35 to 80 cells had to be plated for each infected cell detected. Considering the variability of cell viability and plating efficiency, these results were interpreted to mean that most, if not all, of the cells were persistently infected with NDV. No interferon was detected in the culture fluid from BHKNDV cell cultures. By methods previously described (7), harvested fluids were treated with NDV antiserum and then incubated overnight with control BHK cells. Following challenge with vesicular stomatitis virus (VSV) (MOI = 1.0), BHK cells with and without exposure to fluid from BHKNDV cells were completely destroyed at 48 h and virus yields were comparable (1.0 x 109 to 1.9 x 109 PFU/ml) when assayed in primary chicken embryo (CE) cells. The absence of detectable interferon and the complete susceptibility of BHKNDV cells to direct challenge with VSV is in marked contrast to the findings for mouse L cells persistently infected with the Herts strain of NDV (LNDV). In the case of LNDV cells, 10 to 20 U of interferon are always present in the medium and the cells are completely resistant to superinfection with VSV (7). The BHK-21 cells used for this study contained the R-type virus-like particles reported to occur commonly in this cell line (1, 2). In BHKNDV cells, R-type particles and NDV were not mutually exclusive; both were frequently observed in the same cell (Fig. 1). When the BHKNDV cell line had stabilized 3 months after the infection was initiated, it was noted that the plaque diameter of the carried virus at 37 C in CE cell cultures was <1 to 1 mm after 7 days; at this time wild-type NDV plaques were 2 to 4 mm in diameter. This finding is similar to what has been reported for the virus from LNDV cells (7). Twenty-six virus plaques isolated from BHK NDV culture fluid were tested for efficiency of plating at 37 and 43.5 C, as described previously (3). The 43.5/37 C ratio for six wild-type NDV clones whereas the ratio for the 26 clones was >0.5, from BHKNDV cells ranged from 7.7 x 10' to <7.0 x 10'. Clearly, the persistent infection of BHK NDV cells had spontaneously selected a population of ts viruses, similar to the selection previously recorded with LNDV cells (3). Of the 26 ts virus clones from BHKNDV cells, 17 clones were stable on passage, whereas 9 clones rapidly reverted to the wild type. The 17 stable clones were tested for their RNA phenotype at 43.5 C, using techniques previously described (3). All NOTES clones were defective in RNA synthesis at the nonpermissive temperature: 16/17 synthesized less than 10%; 1/17 synthesized 13% of the RNA made at the permissive temperature (37 C). These data were reminiscent of those previously reported for virus isolated from LNDV cells (3, 4). An attempt was made to establish a persistent infection in the MDCK line of canine kidney cells. The methods employed were identical to those used with BHK-21 cells, as described above. Cells infected with NDVo at all MOI were passaged at 7 to 10 days, although clear cytopathic changes were evident. After this initial passage, the cultures became confluent in from 4 to 30 days with multiple changes of medium. Virus-induced cellular destruction was almost constantly present; one bottle of cells could be split to only two new bottles. By cell passage 7 or 8 (±100 days since infection), the cell lines were completely destroyed. Before this loss of viability of the cells, it was noted that virus was always present in the culture fluid (10' to 106 PFU/ml) and that a high proportion of small plaques ( < 1 to 1 mm in diameter after 4 days at 37 C) was present in the virus population when assayed in CE cells. The proportion of plaque-forming cells and the PFU/hemagglutinin unit ratios were similar to those reported for the BHKNDV cell line. Fluids were sampled from MDCK cells between 60 and 70 days after the infection was initiated (MOI = 0.1 and 0.01) and a total of 24 clones was selected for further characterization; these included 13 small clones (<1 to 1.5 mm) and 11 large clones (1.5 to 4 mm). Wild-type NDVo was also cloned and four small (1 to 2 mm) and three large (2 to 4 mm) plaque progeny were selected for further study. The first passage of these clones in 2-ounce bottles of primary CE cells was tested for ability to destroy CE cells at 37 and 43.5 C; the "welled-tray" technique described elsewhere was used (4). All of the clones selected from wild-type NDVo produced complete cytopathic effect at both temperatures, as expected. Of the 24 plaques selected from persistently infected MDCK cells, the 11 "large" plaque clones produced cytopathic effect at both 37 and 43.5 C, whereas 8 of the 13 "small" plaque clones failed to destroy CE cells at the higher temperature. When these eight clones were tested for their efficiency of plating, the 43.5/37 C ratio ranged from <2.1 x 10' to <1.6 x 106, clearly identifying them as ts mutants. Six of the eight ts mutant clones were stable on passage and all six stable clones were found to have an RNA- phenotype ( < 10% RNA synthesis at 43.5 compared to 37 C). Therefore,

3 1334 NOTES J. VIROL. 44~~~~~~ tv~ : a. 7t X 4f.r~~: 'n~ 4 WP* i r. ~ p K 4ir~~ ~ ~ ~ ~ ~ 4 - ^*,K 2t<<X* ";' 4iEX, i 44,. 40~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4 FIG. 1. Thin-section electron microscopy of BHK-21 cells persistently infected with NDV. Cell monolayers were scraped from the glass, pelleted by low-speed centrifugation, fixed for 90 min in Karnovsky's solution, and postfixed in Dalton's chrome osmium for 2 h. Fragments of fixed pellets were embedded in Spurr's low-viscosity medium and sectioned with diamond knives. Sections were stained with 3% uranyl acetate and Reynold's lead citrate, and viewed through an AEI EM6B electron microscope at 80 kv. Bar is 100 nm. Panel A: single cell showing R-type particles in vesicle (arrow at top) and three budding NDV particles (arrow at bottom). Panel B: note R-type particles in same cell with budding NDV, NDV nucleocapsid material, and altered cell membrane (arrow). ^ i.- Y;..S 1; 4'-- the short-term persistence of NDV in MDCK cells was accompanied by continuous virusinduced cell damage, eventual total cell destruction, and selection of a virus population, one-third of which was ts mutants. Wild-type NDVo populations contain only 1% spontaneous ts mutants (4). The results illustrate several important points regarding persistent infection of cell lines with NDV. First, selection of ts mutants occurs in BHK-21 and MDCK cells persistently infected with this virus. In the case of BHK cells, a long-term persistent infection was established in which ts mutants completely replaced the wild-type virus population. In the case of MDCK cells, it was not possible to establish a long-term persistent infection due to permissiveness of the cells for NDV. Despite frequent

4 4~ ~t,.. VOL. 16, VOL.~~ J : NOE.4,, V8 *,. X~~*&$ 't;k M. *-.' w A $~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 'r^ 4.,,^. b>.0.-s At,-,,s-. B 44,^.,*,F't,'- D V >, *41~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 40~~~~~~~~~~~~~~~~~'. F-7 a~a.$ 4, *, a fl na,~~ feedings and judicious intervals between cell passages, the persistently infected MDCK line was lost about 100 days after initiation of the infection. Even with this degree of permissiveness, 33% of the virus clones isolated at 60 to 70 days were ts mutants. Secondly, all the ts mutants isolated from persistently infected BHK and MDCK cells were defective in RNA synthesis at the nonpermissive temperature, which is in accord with the results in mouse L cells (3-5). This evidence points to the possible importance of a viral transcriptional defect in the establishment of persistent infection (5). Thirdly, in complete contrast to LNDV cells (7), BHKNDV cells make no interferon and are completely susceptible to VSV, suggesting that 4r ~ 1 p:-.a AS C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~j FIG. 1B. b NOTES 1335 interferon mediation is not essential for the establishment or maintenance of the persistently infected state. This investigation was supported by Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases and was sponsored by the U. S. Army Medical Research and Development Command, Washington, D. C., under contract no C7046. The collaboration of John J. Cardamone, Jr., with electron microscopy is gratefully acknowledged. LITERATURE CITED 1. Albu, E., and K. V. Holmes Isolation and preliminary characterization of the RNA-containing R-type virus-like particle of BHK-21 cells. J. Virol. 12: Bernhard, W., and P. Tournier Infection virale inapparente de cellules de hamsters decelee par la

5 1336 NOTES microscopie electronique. Ann. Inst. Pasteur Paris 107: Preble, 0. T., and J. S. Youngner Temperature-sensitive mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. 9: Preble, 0. T., and J. S. Youngner Selection of temperature-sensitive mutants during persistent infection: role in maintenance of persistent Newcastle disease virus infection of L cells. J. Virol. 12: Preble, 0. T., and J. S. Youngner Temperature-sensitive defect of mutants isolated from L cells persistently infected with Newcastle disease virus. J. Virol. J. VIROL. 12: Preble, 0. T., and J. S. Youngner Temperature-sensitive virus and the etiology of chronic and inapparent infections. J. Inf. Dis. 131: Thacore, H. R., anid J. S. Youngner Cells persistently infected with Newcastle disease virus. I. Properties of mutants isolated from persistently infected L cells. J. Virol. 4: Youngner, J. S., A. W. Scott, J. V. Hallum, and W. R. Stinebring Interferon production by inactivated Newcastle disease virus in cell cultures and in mice. J. Bacteriol. 92: