Studies on the Cytopathic Effects of Newcastle

323

J. gen. Virol. 0973), 2x, 323-337 Printed in Great Britain

Studies on the Cytopathic Effects o f Newcastle Disease Virus: the Cytopathogenicity of Strain Hefts 33 in five Cell Types By D. J. A L E X A N D E R

Central Veterinary Laboratory, New Haw, Weybridge, Surrey, U.K. G. H E W L E T T * , P. REEVE* AND G. P O S T E r Department of Virology, Royal Postgraduate Medical School, Ducane Road, London WI2, U.K.

(Accepted 20 June 1973) SUMMARY

The cytopathogenicity and production of Newcastle disease virus (NDV) strain Herts cultivated in chick embryo (CE), baby hamster kidney (BHK-2I), HEp-2, MDBK and L929 cells was investigated. Infection at high multiplicities (IOOO p.f.u.]cell) induced cell fusion in cultures of all cell types within 3 h after infection. Infection at low multiplicities (I o to 20 p.f.u./cell) produced extensive cell fusion in CE, BHK-2I, HEp-2 and L929 cultures within 24 h, but MDBK cells failed to fuse. The latter cells failed to show high levels of virus haemagglutinin at the cell surface or accumulation of virus products, but infective virus was released to significantly higher titres than from the cell types which fused. However, infected MDBK showed similar levels of virus-induced cell damage to the plasma membrane, as measured by the release of lactate dehydrogenase, to HEp-2 and L929 cells which were susceptible to fusion. The morphology of the c.p.e, produced by strain Herts in the different cell types is described. The relationship of virus accumulation and virus release to the process of cell fusion is discussed.

INTRODUCTION

Previous studies with Newcastle disease virus (NDV) strains that differ in the c.p.e. induced in chick embryo (CE) cells and baby hamster kidney (BHK) cells have suggested that c.p.e, in these cells is caused by an accumulation of virus products (Reeve & Alexander, 197o; Reeve, Rosenblum & Alexander, 197o; Reeve & Poste, 1971; Reeve et al. I971; Reeve et al. I972 ). Further, c.p.e, may occur when the rate of production of virus material is greater than the rate of release of virus from the cell (Alexander, Reeve & Poste, 1973). Work with other viruses supports this mechanism of cytopathogenicity (Plowright, I962; Compans et al. I966; Mallucci & Skehel, I97I ; Bablanian, I972). Holmes & Choppin (I966) and Compans et al. (I966) using simian virus 5 (SVs) demonstrated that the infected cell is as important as the virus itself in determining the degree of c.p.e. In infections of monkey kidney (MK) cells, SV5 was non-cytopathogenic, was released * Present address: Department of Bacteriology, University College Hospital Medical School, London, WA., U.K. t Present address: Department of Experimental Pathology, Roswell Park Memorial Institute, 666 Elm

Street, Buffalo,New York 142o3, U.S.A.

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to high titres and virus products did not accumulate, but in BHK 2I-F ceils SV5 was highly cytopathogenic, little virus was released and accumulation of virus products took place. NDV also appears to show degrees of cytopathogenicity depending on the cells infected: our previous studies suggest that c.p.e, occurs earlier and is more dramatic in BHK than in chick cells (unpublished observations). Johnson & Scott (I964) suggested that NDV grown in HEp-2 ceils has an intranuclear phase and produces two morphologically distinct types of polykaryocyte formation. Growth of NDV in mouse L cells is reported as abortive (Reda, Rott & Schafer, 1964; Thacore & Youngner, 1969) in that increases in released haemagglutinin (H.A.) are not associated with comparable increases in released infectivity, although some infective particles are released. Thacore & Youngner (1969) also reported that no c.p.e, occurred if L cells were infected at an input multiplicity of o.I p.f.u./cell. Bader & Morgan (1961) showed that within 48 h of infection, one IDs0 of NDV per culture induced more than IO ~ cellular lysis in HeLa ceils, while a similar level of c.p.e, was induced in L cells only if multiplicities of o'5 to o'7 ID~0/cell were used. However, the study of macroscopic c.p.e, after infection at low multiplicity gives little guide to the effect of virus on the single infected cell, particularly when there is little released infectivity. One of the cytopathic effects of NDV in CE cells is the formation of polykaryocytes, which occurs several hours after the infection of cells with moderate or low multiplicities of cytopathic NDV strains. This type of cell fusion is related to the intracellular growth of the virus and requires virus-specific macromolecular synthesis (Reeve & Poste, 1971 ; Reeve et al. 1971 ; Poste et al. I972c). Polykaryocytes may also be formed after treatment with very high multiplicities of either infective or non-infective virus; this type of fusion is a laboratory phenomenon which occurs within 3 h and does not require host-specific or virus-specific macromolecular synthesis. Bratt & Gallaher (I969) designated these types of cell fusion as 'fusion from within (FFWI)' and 'fusion from without (FFWO)', respectively. In a recent study Bratt & Gallaher (1972) showed that NDV strains which induce polykaryocytes in CE cells by FFWO are equally capable of fusing BHK and MDBK cells by this method. However, although polykaryocytes may be formed in CE and BHK cells by low multiplicities of infective virus, none of the NDV strains that they have tested induced FFWI in MDBK cells even though these cells were infected productively. We report various aspects of NDV cytopathogenicity and growth in CE, BHK HEp-2, MDBK and L cells with a view to demonstrating relationships that may exist between virus growth, accumulation, release and cytopathogenicity.

METHODS

Newcastle disease virus. The highly virulent cytopathic strain Herts 33 (Herts) was grown in eggs (Reeve et al. 197o). Cell cultures. Primary chick embryo (CE) cells were prepared from IO to I2-day-old

embryos (Alexander, r97i ). CE cells, BHK-2I clone 13 (BHK) ceils (original seed culture provided by Dr J'. Best, St Thomas's Hospital Medical School, London S.E.I.), Madin-Darby bovine kidney (MDBK) cells (original seed cultures provided by Mr I. Lukey, Central Veterinary Laboratory, New Haw, Weybridge, Surrey), mouse L-929 cells (original seed cultures provided by Dr L. W. Greenham, University of Bristol) and HEp-2 cells were all grown in Eagle's BHK medium supplemented with IO ~ tryptose phosphate broth and IO ~ foetal calf serum. Cells were infected by virus in Eagle's BHK medium, which contained only 2 ~ foetal calf


Cytopathogenicity o f N D V in five cell types

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serum (maintenance medium). After adsorption for I h at 37 °C the virus was removed, the cells washed once with warm phosphate-buffered saline (PBS), p H 7.2, overlaid with maintenance medium and incubated at 37 °C. Haemadsorption assay. Coverslip cultures were infected with Io to 2o p.f.u./cell of the Herts strain of NDV. At the times specified, coverslip cultures were washed three times in PBS and treated with o'5 ml o'5 ~ (v/v) chicken red blood ceils (RBC) at 4 °C for 2o min. Coverslips were then washed three times in ice cold PBS and fixed in methanol for at least 3o min before staining with May-Grtinwald-Giemsa (Reeve & Poste, I97I). After staining, the RBC adsorbed to cells were counted and expressed as the number of RBC per nucleus. Estimation of fusion as % polykaryocytosis. Fusion from within (FFWI). Cell coverslip cultures were infected with Io to zo p.f.u.]cell of strain Hefts. At the specified times cell cultures were fixed in methanol and stained with May-Gfiinwald-Giemsa (Reeve & Poste, I971). Fusion from without (FFWO). Cell coverslip cultures were treated with Iooo p.f.u./cell for 3 h then fixed and stained as above (Poste et al. I972e). The extent of NDV-induced cell fusion, expressed as ~ polykaryocytosis, was estimated by counting the number of nuclei present in polykaryocytes and expressing this as a percentage of the total number of nuclei present in the same microscope field (Reeve & Poste, I97I). Estimations of virus growth. Confluent monolayers in 9 cm plastic Petri dishes were infected with Io to 2o p.f.u./cell of strain Hefts. After virus adsorption the washed monolayers were covered with 3 ml maintenance medium. At the specified times after infection the medium was removed and released haemagglutinin activity (H.A.) and infectivity in eggs (EIDs0) were determined as described previously (Alexander, Reeve & Allan, I97o), except that the EIDs0 end-point was estimated from tables (Meynell & Meynell, I965). The monolayer was washed twice with PBS and scraped from the dish into I ml of ice-cold PBS. The cells were disrupted by sonication, the debris pelleted at 2ooog for IO min and the H.A. titre of the supernatant fluids estimated. The protein content of the supernatant fluids was estimated by the method of Lowry et al. (195I) and the cell-associated H.A. activity expressed as units (H.A.U.)[mg protein. Cell leakage and lysis was measured by the release of lactate dehydrogenase (LDH) activity into the supernatant fluids of cell cultures. Infected cell cultures in 5 cm plastic Petri dishes overlaid with I ml of maintenance medium were taken at the specified times and the supernatant fluids removed. The cells were then scraped from the dish (microscopic examination showed that only a few isolated cells remained), suspended in I ml of maintenance medium and sonicated to disrupt all the cells. Both the supernatant fluids and the sonicated cell suspension were then assayed for L D H activity by the method of Kornberg (I955). Released L D H activity was expressed as ~ total activity. RESULTS

Virus growth The activity of haemagglutinin released from the cells after infection by NDV strain Hefts differed widely according to the cell type (Fig. I). Up to 8 h after infection none of the supernatant fluids contained detectable H.A. L cell cultures released no detectable H.A. until 20 h after infection and at 24 h had only reached Io H.A.U./ml. H.A. release from HEp-2 cell cultures was similar, being first detectable at


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The release of infective virus from different cell types and its relationship to activity of released haemagglutinin

Table i.

Released virus Cell type CE

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* Released infectivity was estimated 24 h after infection of CE, HEp-2, L and M D B K cells but 2o h after infection of B H K cells.

I2 h and reaching 20 H.A.U./ml by 24 h. B H K cells released H.A. from Io h until 20 h after infection when a maximum level of 80 H.A.U./ml was reached. Cultures of CE and M D B K cells showed much greater H.A. release: CE cells producing 5~o H.A.U./ml by 24 h, while M D B K cells reached I28O H.A.U./ml at 24 h which increased to 5t2o H.A.U./ml at 32 h after infection. Released H.A. and released infectivity were approximately proportional for each cell type with the exception of L cells (Table I). The EID~o/H.A.U. ratio of virus released from L cells was o'7 x io ~ compared with an average of 6.o × io 6 for the other cell types. Cell-associated H.A., expressed as H.A.U./mg protein, was detected at 8 h after infection in M D B K ceils and at 4 h after infection with the other four cell types (Fig. 2). The production of cell associated H.A. followed a common pattern but there were large differences in the rates of production and amounts produced in the different cells.


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Haemadsorption is also a measure of virus production, basically measuring the degree of cell surface modification. The results (Fig. 3), predictably, followed the pattern of production of ceil-associated H.A. It is not practicable to count cultures with more than 30 red blood cells adsorbed per nucleus, for this reason no points past I2 h after infection were included for B H K cells. The degree of haemadsorption to infected L cells tended to fluctuate after the onset of c.p.e, and cell fusion and the average number of red blood cells]nucleus at 2o h was considerably lower than expected (Fig. 3)The very limited haemadsorption to M D B K cells, compared with that for other cells, suggests that the relatively large amount of virus released was cleared very quickly from the cell surface or that the virus was released from foci which were limited both in size and number.

The cytopathic effects The earliest c.p.e, in any of the cells following infection by Io to 20 p.f.u.lcell of NDV strain Herts was the low level of cell fusion in CE, B H K and HEp-2 cells at 4 h after infection (Fig. 4)In monolayers of B H K cells fairly large polykaryocytes were formed by 8 h (Fig. 5b). Under the conditions of this study these polykaryocytes increased in size by fusion of more cells until the polykaryocytes themselves apparently fused to form enormous polykaryocytes 22

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consisting of almost the entire monolayer by I6 h after infection (Figs. 4 and 5c.) These 'massive' polykaryocytes eventually degenerated and peeled from the coverslip. Monolayers of CE cells followed the same course as B H K cells and formed 'massive' polykaryocytes before cell death (Fig. 6a-d), however, polykaryocyte formation occurred at a slower rate in CE than in B H K cells (Fig. 4). The fusion of HEp-2 cells by NDV strain Herts never induced 'massive' polykaryocytes, although large discrete polykaryocytes were formed (Fig. 7)- The progression of fusion in HEp-2 cultures was at a slower rate than in CE cells and about 2o % of the HEp-2 cells did not become involved in syncytia within 24 h of infection (Fig. 4). The polykaryocytes which were formed rarely had more than 30 nuclei and appeared to round up and lyse or detach from the glass before fusion into larger polykaryocytes could take place (Fig. 7 a-d). In some polykaryocytes the nuclei were grouped towards the centre, while in others, particularly in the earliest hours after infection they were more peripheral. The onset of fusion in L cells was quite dramatic (Fig. 4)- Until I2 h after infection little or no syncytial formation could be seen (Fig. 8 b). However, between I 2 to ~6 h of infection of L cells 6o % of the nuclei became involved in polykaryocytes and by 2o h after infection fusion of the whole monolayer had taken place (Fig. 8 d). In infections of the other cell types no gross changes were noted in the nuclei, although during later stages of infection HEp-2 nuclei took up more stain and became less distinct. The nuclei of infected L cells showed marked morphological changes and by 2o h after infection were considerably damaged. Monolayers of M D B K cells infected with IO to 2o p.f.u./cell of strain Herts did not form polykaryocytes, although by 24 h after infection the cells appeared less healthy and stained less distinctly (Figs. 4 and 9a, b). Cell death in infected M D B K cells did occur and was apparently due to lysis of individual cells without rounding-up (Fig. 9 c).


Cytopathogenicity o f N D V in five cell types

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Fig. 4. Polykaryocyte formation in BHK (0), HEp-2 (A), L (A), CE (O) and MDBK (11) cells infected with io to 20 p.f.u./cell of NDV strain Herts.

Fusion from without Infection of all five cell types with xooo p.f.u./cell of N D V strain Hefts induced cell fusion within 3 h (Fig. IO). Compared with FFWI, monolayers of B H K and CE cells showed much lower levels of polykaryocytosis (47 and 46 ~ of nuclei involved, respectively). The polykaryocytes were also relatively small although they often contained 15 to 2o nuclei (Fig. Ioa, b). About 4o ~ of the nuclei in L cell cultures were involved in polykaryocytes after FFWO. The polykaryocytes were small, usually involving three to ten nuclei although some of ten or more nuclei could be seen (Fig. Ioe). Polykaryocytes in HEp-2 cells contained 3 to IO nuclei and involved about 48 ~ of the total nuclei. The polykaryocytes formed were quite distinctive in that the nuclei were found almost exclusively at the periphery of the polykaryocytes (Fig. Iod). Monolayers of M D B K cells showed FFWO, in contrast to the ability of the virus to induce FFWI in these cells, and up to 26 ~o of the nuclei were involved in polykaryocytes. This level of polykaryocytosis is comparable to our previous estimate of 32 ~ using 2000 EIDs0]cell of inactivated strain Herts (Poste et al. ~972c). Few polykaryocytes in M D B K cultures contained more than six nuclei, but large numbers of small polykaryocytes containing 2 to 5 nuclei were present (Fig. Ioe).

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Fig. 9. Figs. 5 to 9. Different cells (Fig. 5, BHK; Fig. 6, CE; Fig. 7, HEp-2; Fig. 8, L'cells; Fig. 9, MDBK) infected with io to 20 p.f.u./cell NDV strain Herts at various times after infection. (Magnification

x 66).

Release of lactate dehydrogenasefrom infected cells Increase in L D H activity in the supernatant fluid of cells infected with NDV has been used in this study as an indication of cell leakage and lysis. After infection the leakage of L D H from the different cell types generally followed the pattern seen previously for cell associated H.A., haemadsorption and cell fusion (Fig. I i), although increases in L D H release occurred later after infection than increases in these other activities. The L D H activity was not released at a high level in the supernatant fluids of L cell monolayers until 28 h after infection; this later release may have been due to cells detaching from the glass but not lysing until some time later. Monolayers of M D B K cells showed relatively high levels of released L D H activity at 24 h after infection, when cells appeared comparatively healthy (Fig. 9 b); this suggests that these cells were particularly 'leaky' prior to lysis and death. Control cells run in parallel showed small rises in extracellular L D H activity but in no cell type did it exceed lo % of the total by 24 h.


Cytol:athogenicity o f N D V in five cell types

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Fig. IO. Cells infected with NDV strain Herts at IOOOp.f.u./cell and fixed 3 h after infection. (a) BHK cells; (b) CE cells; (c) L cells; (d), HEp-2 cells; (e) M D B K cells. (Magnification × 66).

DISCUSSION

The effects of NDV strain Herts on the different cell types reported in this study are similar to those reported previously in work with other cytopathic NDV strains. Bader & Morgan 0 9 6 0 showed that NDV was cytopathic in L cells when relatively high input multiplicities were used. Our results, showing the released and cell-associated virus in infections of L cells, are similar to those of Redaet al. 0964). The abortive cycle of NDV in L cells has been described (Reda et al. 1964; Thacore & Youngner, 1969).


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Fig. I I. Extracellular lactate dehydrogenase (LDH) activity in cell cultures. 0 - - O , BHK; O--©, CE; A--A, HEp-z; 2x--~, L; i - - i , MDBK cells infected with NDV strain Herts. The activity in the extracellular medium is expressed as a percentage of the total cellular activity. Absolute values of total activity per culture were: BHK, 4o0 mU; CE, 36o mU; HEp-2, z450 mU; L, 545 mU and MDBK, 372 mU. In our study we report two morphologically distinct types of polykaryocyte similar to those reported by Johnson & Scott (1964) after infection of HEp-a cells with NDV. These two forms consist of those w i t h ' peripheral' o r ' central' nuclei. T h e ' peripheral' nuclei-type polykaryocyte predominates earlier after fusion and is somewhat similar to syncytia caused by F F W O . It is likely that the different forms are ' y o u n g ' and ' o l d ' syncytia. Johnson & Scott ([ 964) used very low multiplicities of virus and were unable to confirm their suggestion that this was the case. Degeneration of the nuclei in L cells and, to a much lesser extent, H E p - e cells was more marked than in the other cells and may reflect the reported accumulation of antigen in the nuclei of these cells (Johnson & Scott, 1964; Reda et al. 1964). Bratt & Gallaher (1972) showed that cytopathic strains of N D V that fuse CE or B H K z I-F cells from within do not fuse M D B K cells, although these cells were both productively infected and at 20 h after infection were reported to show other cytopathic changes. Similarly, it has been shown that SV5-infected M K and M D B K cells release virus to high infectivity and do not fuse, but SV5-infected BHK21-F cells accumulate virus, fuse and die (Compans et al. 1966; Holmes & Choppin, 1966; Klenk & Choppin, 197oa ). In this study we have examined cell fusion and cell lysis and death as two measures of cytopathogenicity. Previously we considered these to occur by a similar mechanism, since all N D V strains that induced F F W I were cytopathic for CE and B H K cells and virulent for chickens and eggs (Reeve & Alexander, I97o; Reeve & Poste, 197I; Poste et al. I97ZC).


Cytopathogenicity o f N D V in f i v e cell types

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The present results do not detract from the possibility that whatever mechanism brings about fusion also causes cell death. Thus infected BHK cells accumulate virus products at a greater rate than CE cells and fuse and die earlier than CE cells. However, the failure of infected MDBK cells to fuse from within, despite their ability to fuse from without, suggests the possibility that two distinct mechanisms may be involved, one causing fusion and the other death. This is particularly emphasized by the occurrence of cell leakage and death at similar rates in both fused and non-fused cell types following infection with NDV. It is known that polykaryocyte formation does not lead to the death of fused cells as rapidly as they have died in this study, although this may vary depending on the cell type and, more importantly, on the size of the polykaryocyte (Harris, I97o; Poste, I972). If two mechanisms of virus-induced cell death are involved it may be that the]high rate of release by MDBK cells is responsible for a high level of membrane damage and eventual death. The fast clearance of virus from MDBK cells may also be related to the lack of polykaryocyte formation (see below). Explanations of why one type of cell should fuse after infection but not another have been offered by other authors. Bratt & Gallaher 0972) discussed the possibility of a hypothetical 'fusion factor' which was missing in virus infections of MDBK cells. Klenk & Choppin (197o a, b) suggested that variations in the composition of the cell membrane may be responsible for the differences in the fusing ability of the paramyxovirus SV5 in different cell systems. Fusion of cells from within following virus infection may be caused by modification of the cell surface membrane due to the accumulation of virus components (Ejercito, Kieff & Roizman, 1968; Keller, Spear & Roizman, I97o). Certainly NDV strains which accumulate virus products also bring about cell surface changes and fuse cells (Alexander et al. I97o; Reeve et al. I97o; Reeve & Poste, I97I ; Poste et al. I972b; Reeve et al. I97z; Alexander et al. I973). The present results also add evidence that fusion of cells is brought about by accumulation of virus products. Monolayers of CE, BHK, HEp-2 and L cells infected by NDV reached high levels of cell-associated H.A. or haemadsorption and fused, whereas infected MDBK cells which showed little or no cell-associated H.A. or haemadsorption did not fuse. The levels of cell-associated H.A. in HEp-z or L cells were much higher than the levels of released H.A. but were low compared with cell-associated H.A. in CE and BHK cells. This suggests that, if fusion is related directly to accumulation of virus products, different cells may have different thresholds of accumulation above which they fuse. Conversely, the levels are different and may indicate that cell fusion occurs in the absence of accumulation of virus products, The theory that differences in cell membrane composition are responsible for differences in cell fusing ability is particularly attractive since it can account for variations in accumulation of virus products as well as in fusion. Klenk & Choppin 0969, I97oa) showed that MDBK and primary rhesus monkey-kidney (MK) cells have a higher cholesterol/phospholipid ratio than BHKzr-F cells or hamster kidney (HaK) cells and suggested that this may be responsible for differences in fusing ability. However, Poste et al. 0972a), using a wide range of viruses and cell types, failed to confirm this hypothesis. It is not known if fusion from without and fusion from within are caused by the same mechanism. If FFWI is due to extensive modification of the cell membrane by accumulation of virus products then very high multiplicities of virus may bring about similar modification by virus attachment and fusion, as described by Apostolov & Almeida (1972) with Sendai virus. Haemadsorption studies suggest that, after infection with levels of virus that induce


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P. REEVE A N D G. POSTE

FFWI in other cells, M D B K cells are modified only at isolated places on the cell surface. Under these conditions insufficient cell surface modification may occur to induce FFWI, but when high multiplicities of virus are used modification over a larger area is induced. In this study not only were different amounts of virus accumulated and released in different cell types, but there were considerable variations in the total virus production. A further consideration in understanding the cytopathogenecity of a virus is that the effect of that virus on a cell may also be modified by the metabolism of that cell, as work with coxsackie B virus has demonstrated (Khesin & Amchenkova, ~972). Some of this work was done while D. J. A. and G. H. were supported by the Agricultural Research Council (A.R.C.) as members of the Virology Department, Royal Postgraduate Medical School, London, and the Bacteriology Department, University College Hospital Medical School, London, respectively. P. R. was supported by the Wellcome Trust and G. P. by the Cancer Research Campaign. This research was aided by a grant from the A.R.C. We thank Dr M. A. Bratt for preprints of his recent papers.

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(Received 8 March I 9 7 3 )
