Evaluation of suspended chick embryo lung cells as a host system for influenza virus

VIROLOGY

4, 109-125

Evaluation

NADA Division School

(1957)

of Suspended a Host System LEDINKO~,

of Virology, of Medicine,

VICTOR

Chick Embryo Lung for Influenza Virus’ V. BERGS,~

AND

WERNER

Cells

as

HENLE

The Department of Public Health and Preventive Medicine, University of Pennsylvania and The Children’s Hospital of Philadelphia, Pennsylvania 9ccepted

May

S, 1967

Inoculation of suspensions of trypsinized chick embryo lung cells with the WSE strain of influenza A virus yielded results comparable in many respects to those obtained with other strains in the allantoic cavity. The degree of adsorption of seed virus onto the cells varied inversely with the amount inoculated from 60 to 90%. Adsorption was prevented by prior exposure of the cells to receptor-destroying enzyme (RDE). On addition of RDE after infection with small doses of virus one-step growth curves were obtained whereas after large doses the yield of hemagglutinins was not affected but they became detectable in the media earlier than in the absence of the enzyme. Noninfectious hemagglutinins were found in increasing proportions in the progenies as the inoculum was increased from 1 to 30 EID,o per cell, which appeared to be the point of saturation. Inocula providing less than 1 EIDso per cell yielded dominantly infectious virus. The system did not lend itself, however, to accurate quantitative analyses of cell-virus interrelatiorlships in that a number of observations suggested that possibly not more than one in ten of the available cells is capable of supporting propagation of the virus. INTRODUCTION

Much information has been obtained regarding the actions in the chick embryo-influenza virus system. ation of the exact quantitative interrelationships was by the fact that the number of available susceptible mated only within limits. It was hoped, therefore, that

host-virus interHowever, evalupartly hampered cells can be estia tissue culture

1 The work described in this paper has been supported by a grant-in-aid from the National Institutes of Health, United States Public Health Service. 2 Public Health Service Research Fellow of the National Heart Institute. Present address: Public Health Research Institute of the City of New York, Inc., New York 9, New York. 3 Predoctorate Fellow, United States Public Health Service. 109

system could be developed which would be more readily applicable to quantitative study of such problems as incomplete virus formation, interference, multiplicity reartivat’ion, and genetic recombination. Cultures of HeLa cells and other human cell strains upon infection with eggadapted influenza virus were found to he incapable of supporting the production of infectious virus, but they readily yield noninfectious hemagglutinins (Henle et al., 1955; Girardi et al., 1956; Deinhardt and Henle, 1957). Tissue cultures of chick embryo hmgs, on the other hand, were shown to produce infectious virus (Pearson and Endcrs, 1!)11; Tyrrell, 3955; Granoff, 1955; Ledinko, 1955). In the present study the potential usefulnessin quant’itative studies of suspensionsof t*rypsinized rhick embryo lung cells was explored. MATJJRIAI,S

AN I) iMICTHOIX3

Vi7-US

The highly egg-adaptfed WSE strain of influenza A virus was used t’hroughout.* Standard seeds, which consist’ed almost entirely of infectious virus, were prepared by allant’oic inoculation of 11-day old chick embryos with lo3 to lo4 $X1150.The allantoic fluids were collected after incubation at 3Gto 37” for 18 to 24 hours. These were used either direc+Jy or after storage in glass-sealedampules in the dry ice chest,. Virus Assays The method of infectivity t,itrations in the chick embryo has been described (Henle, 1949). The test materials were diluted serially in lo- or 3.16fold steps and eight to ten eggs were used per dilution. Infectivity end points (13111so) were calculated, according t’o Reed and ~luench. liar hemagglut,inat.ion assays a method was employed whicbh was about 1 t,imes more sensitive than that previously used (Hcnlc and Henle, 1919). Serial two-fold dilutions of the tissue culture mat,crials were made in l-ml volumes and 0.2 ml of a 0.5 % suspensionof chicken red cells was added to each tube. The HA titer was taken as the highest dilution showing partial though definite agglutination. In the growth curve experiments t,he materials were incubated with equal volumes of suitably diluted receptor destroying enzyme (RDE) of Vibrio cholerae (Finter et al., 1954; Tyrrell, 1955) in order to destroy remaining inhibit’ors of agglutination. In these cases the dilutions were made in * It was kindly stitute, Melbourne,

supplied by Awtraliit.

1)~. Eric

French

of the

Walter

and

Eliza

Hall

In-

INFLUENZA

WRITS

IN

CHICK

EMBRYO

LUXG

CELLS

111

2.5 % sodium citrate saline solution. On account of the more sensitive HA titration technique used here the EID,,/HA ratios of standard virus were of the order of 105.7 instead of 106.3 as previously reported. Receptor-Destroying The preparation?

hkzyme

(RD)

Contained approximately

1280 units per milliliter.

Cell Suspensions Lungs were removed from forty tJo eighty l&day old chick embryos, washed onre with Earle’s balanced salt solution (BSS) and pressed through a syringe into BSS using 1 to 2 ml per one set, of lungs. The tissue was allowed to settle at room temperature. The supernates cont#aining red cells and cellular debris were discarded and replaced by the same volumes of 0.5% t,rypsin$ in BSS. The mixtures were pipetted up and down until the tissue was finely dispersed, incubat’ed at 36” for 30 to 45 minutes and again pipetted until no cellular clumps were observed. After centrifugation at 1000 rpm for 5 to 10 minutes the cells were washed t’wice in BSS. Prior to the final centrifugation the suspensions were allowed to stand at room t,emperature for 5 to 10 minutes to permit, settling of remaining aggregates and the bottom 1 t,o 2 ml were discarded. The cells were finally resuspended in a medium referred to as CM, containing 20 % chick embryo extract (made as a 30 % suspension) and 80 % Scherer’s maintenance solution. The cells were counted in a hemocytometer and the volumes adjusted by the addition of sufficient amounts of CM t)o achieve the desired cellular concentration. For virus studies, 5-ml aliyuots of infected cell suspensions were placed into 25-ml Erlenmeyer flasks, tightly stoppered, and incubated at 36”. Unless ot8herwisc stated, only the flu;d portions of the cult,ures were used for assay of virus activit*y. Other technical deta$s are described in the text. Chick

Embryo

Lung

XXPERIMENTAL

Preliminary experiments indicated that t#he concentration of cells to be employed was critical in that too large a number per milliliter decreased the yield of virus possibly because of extensive acid production and other factors affecting the functioning of the cells. A concentration of about 1 to 2 X lo7 cells/ml appeared to be optimal and such suspensions were employed in the following studies. t It was obtained 1 From Nut,ritional

from the Behring Biochemicals

Werke, Co.

Marburg,

Germany.

112

LEDINKO,

The Adsorption Infection

of Virus

BERGS,

AND

HENLE

onto Lung Cells at Different

Multiplicities

of

Varying amounts of virus were added to cell suspensions to give a range of 0.002 to 850 EIDbO/cell. After an adsorption period of 30 to 45 minutes at 36” the cultures were centrifuged at 2000 rpm for 20 minutes and the EID,, titers of the supernates were determined. The combined results of several tests are shown in Fig. 1. The degree of adsorption gradually increased over the range studied from 60 to about 90% with a decrease in the amount of virus added. Although the accuracy of virus titrations in chick embryos is limited, with the technique employed a difference in titers greater than 0.3 log,, units is considered significant (Knight, 1944). The data suggest that single cells may adsorb certainly more than 100 EIDSO and possibly as many as 500. Horsfall (1955) computed t’hat one entodermal cell of the allantois adsorbs at least 44 influenza B (Lee strain) and 89 influenza A (PRS) virus particles. In these cases, however, only the part of the cell facing the allantoic cavity is available for adsorption. In this regard it is of interest to note that Fogh (1955) found that monkey renal cells are able to adsorb more than 1000 plaque forming units of poliomyelitis virus.

1000

100

10

Saad FIG.

relation

1. The degree to t,he number

1

0.1

0.01

0.001

EIDso/&LL-

of adsorption of virus onto chick of RIKISo added t,o the cultures.

embryo

lung

cells

in

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EMBRYO

LUNG

113

CELLS

Prevention of Adsorption of Virus onto Lung Cells Previously Exposed to RDE Experiments in intact (Stone, 1948) and deembryonated chick embryos (Burnet and Lind, 1953; Finter et al., 1954) have shown that receptors for influenza virus can be removed from susceptible cells by the action of RDE. The cell receptors may subsequently regenerate (Stone, 1948; Finter et al., 1954), but if the relationships between the doses of RDE and virus are properly balanced, one-step growth curves may be obtained in deembryonated eggs @inter et al., 1954). The experiments described below and in subsequent sections were undertaken in order to determine whether infection of chick embryo lung cells can similarly be controlled by RDE. Mixtures of lung cells (0.9 to 1.5 X lo7 cells/ml) and various concentrations of RDE were incubated with frequent shaking at 36” for 1 hour. The original RDE preparation contained 1280 units per milliliter. WSE virus was added to provide from 0.7 to 32 EID~o per cell. After an adsorption period of 30 minutes the cultures were centrifuged, the supernates were titrated for infectivity and the percentage of virus adsorbed was calculated. As shown in Fig. 2, exposure of the cells to concentrations of RDE greater than 2 % (> 25 units/ml) completely prevented adsorption of WSE virus when added subsequently. A concentration of 0.5% RDE or less had little or no effect. A few experiments with monkey renal cells gave principally similar results.

0

2

4 Per

FIG.

with

2. Prevention RDE.

of adsorption

6

cmt

of seed virus

8

10

RDE by

prior

treatment

of the

cells

114

LEDINKO,

The &j’ect of Addition

BERGS,

ASD

HESLE

of RDE after Infection

of Lung C’dls

Suspensions containing 0.65 to 1.3 X lo7 cells were mixed with virus to provide from 0.01 to 34 EIDbo per cell and incubat’ed with frequent shaking for 30 minutes at 36”. RDE was then added to a final concentrat’ion of 6 % (75 units/ml) and the mixtures were incubated for an additional hour with repeated agitation. Thereafter the cells were washed four times with ice-cold BSS, containing one-fourth the concentration of bicarbonate, and aliquot#s were resuspended in medium cont’aining no or varying concentrations of RDE. After further incubat,ion for 20 hours at 36” t,he hemagglutinin titers were determined and the results are shown in Table 1. When the inowlum contained more t’han 1 EIDso per caell (Expts. 1 and 2) the RDE treatment’ had no significant effect, on hemagglutinin product’ion presumably because all susoept’ible cells had been infect’ed prior to the addition of the enzyme. Following inoculation of less than 1 EIDso per cell (Expts. 3 and 4) the yields of hemagglutinin were reduced by the action of RDE and the degree of redu&ion increased with the concentration of the enzyme in t’he medium. When the RDE was removed after 1 hour, second infectious cycles presumably occurred because maximally at’tainable HA titers mere obtained. These results are essentially in agreement wit,h similar data obtained in deembryonat,ed eggs (Finter et al., 1954). They indicate t,hat (a) cell receptors may regenerate as t,he RDE, present’ in the medium, conceivably loses in TABI,

1

HA units/ml Experiment number

Inoculum EIDsi,!cell

Cells ‘ml

Time inyx~aix

of 74, RDE 10

<8 2.1 0.13 0.01

6.5 ~

X 8 x

1.3

x

lOa 106 10'

20 2 20 2 20 2 20

3% 128 1

in Medium 5

2

0

<8

<8

<8

<8

1536

1L280

1536

11536

1536

<8 1280 <8 221

<8 1536

<8 1792

<8 1536

<8 448 <4 192

<8 568

<8 1792 <8 1792

<4 1536

<4 2560

<-l 61

<4 448

-

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115

activity during incubation at 36”; or (1)) at, higher concentrations the enzyme exhibits possibly some toxicit,y for the cells although infec*tion with larger doses of virus revealed no such effects sinc*e the yields of hemagglutininx were not reduced. The Eflect of RDB

on the Latent

Period

It was shown in the preceding section that under conditions where the EIDso in the inoculum exceeded the number of available cells subsequent’ addition of RDE did not affect the total yield of HA in 20 hours of incubation. However, RDE reduces the latent period. A representative experiment is described below. A suspension of 1.2 X 10’ cells/ml was infected with WSE virus to provide 6 EID60 per cell. After 30 minutes at 36” the suspension was divided into two aliquots. To one, RDE was added (6%); the other served as control. After 1 hour of incubation at 36”, both aliquots were washed four times with ice-cold BSS. The control cells were resuspended in RDE-free medium. The cells which had been treated with RDE were divided into two parts, one was resuspendedin medium again containing 6% RDE, the other inmedium without enzyme. The time required for the washing procedures was disregarded since the manipulations were carried out at low temperatures. Samples were withdrawn from each series at various intervals during a total incubation period at 36” of 22 hours and assayed for HA activity. The results are shown in Fig. 3. As seen, the INOCULATION (6 EV&,/CELLI

CELLS

I

WASHED

4 x

..i

2

Y

6

Hours FIG. 3. The effect of RDE added after detection of hemagglutinins in t,he media.

8

10

aftar tnfectior. infection with

22

-6 II:Il),~

per cell on early

116

LEDINKO,

BERGS,

AND

HENLE

RDE-treated cells which were maintained in medium containing enzyme showed the earliest appearance of HA in the medium, the RDE-treated cells resuspended in medium without enzyme followed next, and the control series was last. The latent periods were 2%, 3, and 3% hours, respectively. There were no significant differences in the rates of release nor in the ultimate titers attained in the three series. It has not been determined whet,her the results were caused by earlier release of virus by action of RDE, or whether the enzyme merely prevented readsorption of released virus ont,o the cells on account of recept,or destruction (Cairns, 1955). Growth

Curves of W&Y

Virus

in Lun.g Cells

One-step growth curves can be obtained readily when all cells are immediately infected by the inoculum. On the other hand, when smaller inocula are used the infectious process must be restricted to a single cycle by protection of the remaining noninfected cells. This has been achieved in the chick embryo or in deembryonated eggsby either induction of interference in the remaining susceptible cells (Henle et al., 1947) or by removal of their receptors with RDE (Finter et al., 1954; Henle et al., 1954). Although the latter method, as pointed out above, is not, without uncertaint’ies, one-st’ep growth curves were obtained by this means. Suspensions containing 1 to 2 X lo7 cells/ml were inoculated with WSE virus to provide 6.2 and 0.15 EID60 per cell, respectively. J’ollowing an adsorption period of 30 minutes at 36”, 6% of RDE by volume was added. One hour thereafter the cells were washed four times in icecold BSS (the time required for this procedure was discounted) and resuspendedin medium containing 10% RDE. Aliquots were removed at various times during further incubation for a total of 12 hours. The EID50 and HA titers were determined and the results are shown in Fig. 4. The infectivity titrations revealed that washing had failed to remove all excess seed virus. The EIDr” levels remained constant for 1 to 1% hours. At this time, or 2% to 2% hours after infection, the titers began to rise sharply to reach ultimately the same levels in both series. That the titers reached equal levels was accidental, as will be apparent from the data preserued in the next section. With the larger inoculum (6.2 EIDao per cell) releaseof hemagglutinins became detectable at the same t,ime and the rate of liberation was of an order similar to that of infcatious virus. ,Maximal EIT>so and HA tit,ers were at,tained in 6 and

INFLUENZA

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CELLS

about 8 hours, respectively. The EID,,/HA ratios were 104J on the average, indicating that the progeny of multiply infected cells contained a large proportion of noninfectious hemagglutinins in accordance with similar results obtained in chick embryos (von Magnus, 1951, 1954; Fazekas de St. Groth and Graham, 1954; Finter et al., 1955). Following inoculation of the smaller quantity of virus (0.15 EIDjo per cell) release of HA became detectable only after 5 hours. The EIDbo/HA ratios were 105.6 on the average; i.e., of the order of those of fully infectious (standard) virus. Both the EIDm and HA titers reached a plateau in about 8 hours and no further increases were noted during the experimental period. Thus, it is evident that the addition of RDE after infection limited production of virus presumably by prevention of second infectious cycles. l@‘ect of Multiplicity of Infection on Incomplete Virus Production The experiment, presented in Fig. 4 had shown that the progeny derived from multiply infected cells revealed an EID,o/HA ratio conINOCULATLON J

CE+s

WASHE

4x

4

6 &or

I8 10 infection-

virus

after

inoculation

It---I2 -Hours FIG. 4. Growth curves and 0.15 EIDbo per cell.

of WSE

I12 of the cultures

with

6.2

siderably lower than that derived from singly infected cells; i.e., the former contained appreciable quantities of noninfectious hemagglutinins. The relation of t’he dose of seed virus to the degree of incomplete virus formation was analyzed further, using the same techniques as described in the preceding section. The cell suspensions were infected with varying amounts of standard virus to provide for 0.1 to 40 EIDso per cell and the media mere collected 7 hours later for EID60 and HA titrations. The EID,,/HA ratios of the standard virus inocula averaged 105.*. The data of the various experiments are summarized in Pig. 5. As can be seen the yield of HA increased in proportion t.o the amount of virus inoculated not only when less than 1 EIDLO was added per cell but up to the point when the EIDGO/cell ratio reached about 8. With a further increase in the inoculum the additional yields of HA fell off and a point of saturation apparently was reached when about 30 EIDro were added per cell. Since incubation periods were limited to 7 hours, maximal yields of HA were not yet attained in all instances, as was observed also in the chick embryo (IGnter et al., 1955). Thus, the result,s may denot#e merely that as more and more virus parMes enter individual cells the rate of HA production increases but not necessarily t,he quantities of HA which are ultimately

FIG.

yields

Seed

EID&aI1

5. The relation between the number of infectious virus and noninfectious

(Log)

-

of EID,, inoculated hemagglutinins.

per cell

and the

INFLUENZA

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formed per infected cell. The EIDSo titers increased in parallel with the HA levels at the lower levels of infection. However, when the EID50 in the inocula exceeded the number of cells, the yields decreased again gradually until they leveled off when 30 or more EIDSo were added per cell. It is evident that progenies with EIDEo/HA ratios of standard virus (about 105.8) were obtained when less than 1 EIDso was inoculated per cell, whereas, the ratios in the yields fell gradually to 103.6 as the number of seed EIDb, per cell was increased from 1 to more than 30. The Yield 0.f Virus Per Cell In the above experiment suspensions containing in the order of 107 cells/ml were used. These yielded optimally 103,4 HA units/ml or 0.00025 units/cell. This value is low in comparison to the yields obtained from entodermal cells of the allantois or from HeLa cells (Henle et al., 1955; Girardi et al., 1956). Furthermore, in the cited reports, a less sensitive technique for HA assay was used. However, in the present study only the medium was analyzed from which the cells were removed by centrifugation and it is possible that cell extracts would have increased the yield of HA as observed with infected HeLa cells (Henle et al., 1955). To analyze the situation further a number of additional experiments were carried out. The effect of cell concentration on HA production. In these experiments chick embryo lung cells as well as monkey renal cells were employed since it was found that the WSE strain produced plaques on monolayer cultures not only of the former (Ledinko, 1955) but also of the latter type of cells (Bergs and Ledinko, unpublished data). Chick embryo lung cells were prepared in the usual way. Suspensions of monkey renal cells were prepared by trypsinization of mature sheet cultures.* The latter preparation may represent, therefore, selected cells capable of propagation in tissue cult’ure. The suspensions of the two types of cells were inoculated with sufficient WSE virus to provide between 5 and 10 EIDBO per cell. After incubation at 36” for 30 minutes the cells were washed three times, resuspended in medium, and counted. Thereafter, serial two-fold dilutions of the cells were made in 5-ml volumes and these were incubated for 20 hours prior to hemagglutinin assays. The combined results of several experiments are shown in Figs. 6a and 6b. The yields of HA were related, within limits, to the number of cells present in that they increased up to a critical cell concentration roughly * Obtained

from

the Microbiological

Associates,

Inc.

120

LEDINKO,

Ll-I 3l.2

BERGS,

AND

LA-r-l-r-+--Yl-

7-l-I7I12.8 3.2 ---Cells/ml

HENLE

0.8

2.56

0.2

064

0.16

0.04

0.01

x 106 -

FIG. 6. The effect of rell concentration

on hemagglutinin

production.

in proportion to their number. Yet, the slope of the line reflecting this relation was somewhat steeper than 0.3 as expected with the dilution scale used; i.e., with two-fold greater concentrations of cells slightly more than twice the number of HA units were produced. As the number of cells was raised beyond the critical point the yields fell off rapidly. This decline may be ascribed to unfavorable conditions for cell survival such as production of increasing quantities of acid and insufficient nutrient material in the media. The HA titers maximally attained were similar in both groups (103.’ to 103.5in different experiments). However, the concentrations of the two types of cells required to yield these levels differed by a factor of more than 10. Whereas from 6 X IO6to 2 X lo7 chick lung cells per milliliter gave optimal yields, only from 5 to 8 X lo6 monkey renal cells per milliliter were required to produce similar results. Thus each chick lung cell produced on the average only about >{o the amount of virus as did the monkey renal cell. This may mean that roughly only one in ten of the lung cells was capable of supporting viral multiplication. An attempt was made to answer t’his que&on in the following sets of experiments. Cells were inoculated with either 0.1 to 0.5 or 4 to 7 EIDb,, per cell. After adsorption, the cells were treated in the usual way with RDE and washed. Counts of viable cells were obtained, as determined by lack of staining with trypan blue according to t’he procedure of Girardi et al. (1956). Serial tenfold dilutions of these suspensionswere inoculated into eggs to determine the EIDso. For control purposes the cell suspensions were centrifuged and the titers of extracellular virus present in the suspending medium were determined. These were always found to be significantly lower than the titers of the washed cell suspensions.On the assumption that one infected cell would cause infection in t,he egg, the

INFLUENZA

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expected EIDho was calculated for each cell suspension. From a comparison of the expected EIDM and the EIDro actually obtained, the fraction of cells producing infection in eggs could be calculated. This was found to vary from 0.02 to 0.18 with an average of 0.09, regardless of the number of EIDso inoculated per cell. In some experiments cultures of monkey renal cells were substituted for eggs and the titers were determined by cytopathogenicity. These yielded similar results. These experiments again indicate that only approximately one in ten cells carried infectious virus under the experimental conditions used. DISCUSSION

The results obtained on infection of trypsinized chick embryo lung cells with the WSE strain of influenza A virus agreed principally with those observed with other influenza A strains in the allantois of chick embryos. The extent of adsorption of seed virus varied from 60 to 90 % depending on the quantity added. Adsorption could be prevented by prior treatment of the cells with RDE. Addition of this enzyme to cultures after infection was without effect on the ultimate yields when sufficient virus was added initially to provide more than 1 EIDK, per cell, although hemagglutinins became detectable in the medium after shorter incubation periods than in the absence of the enzyme. This latter finding may be due to the prevention of adsorption of progeny to remaining receptors on already infected cells or to more rapid liberation of virus from the cells. When RDE was added to cultures inoculated with less than 1 EIDW per cell it prevented the occurrence of second infectious cycles and one-step growth curves were obtained, provided the dose of enzyme was properly adjusted. If the infecting dose of virus was large, predominantly noninfectious hemagglutinins were formed in that the EIDEo/HA ratios decreased to values as low as 103.‘j.With small inocula mainly infectious virus was produced having EID,,/HA ratios of lo5 .6 to 105.8.Since a 4-times more sensitive HA technique was employed in the present studies the ratios obtained correspond to values of 106.2t,o 106.4determined for standard virus previously. In preliminary experiments it was also noted that ultraviolet-inactivated WSE virus interfered with the propagation of active challenge virus added subsequently to the culture. While these results indicated that the chick embryo lung suspensions might be useful for study of interactions with influenza virus the quantitative aspects of the system were not as encouraging. The yields of HA per cell were significantly smaller than in monkey renal cultures or the

122

LEDIA-KO,

BERGS,

AND

HESLE

allantoic entoderm, indicating that either only about J~o the amount of virus was produced in lung cells than in the others, or that roughly only one in ten lung cells was participating in the infectious process. Injection of chick embryos with graded numbers of inoculated and washed lung cells, likewise, suggested that at most 10% of the cells were infectjous. Interpretation of these results is complicated, however, by the following considerations. (a) Some of the infectious virus found might, in fact, represent superficially adsorbed seedvirus which had not been removed from the cell surface by RDE treatment,. (b) The infectious processin the cells might have been in eclipse phase at the time of injection and t,hus infectious progeny would have resulted only if the lung cells remained viable in the allantoic cavity, if indeed they all were viable at the start in spite of general lack of staining with trypan blue. (c) The suspensions certainly contained mixed populations of cells (indeed their sizes varied over a considerable range) and thus it is conceivable that aside from fully susceptible cells they contained somewhich were insusceptible and others which were capable of producing only noninfectious hemagglut)inins as in the case of HeLa cells (Henle et al., 1955; Girardi et al., 19.X). Disregarding these uncertainties and assuming that only 10% of the cells were actually involved in the infectious process, the number of HA units produced per infected cell would be 0.0025 on the average (or 0.0006 on the basis of the HA technique used previously). The yields of infectious virus within t’he limited incubation periods studied would average about 100 and lessthan IO EIDso under conditions of infection at low and high seed EIDbU/cell ratios, respectively. With these corrections the results would be in line with those recorded for HeLa or entodermal cells of the allantois (Henle et al., 1947; Fazekas de St. Groth and Cairns, 1952; Henle et al., 1955; Horsfall, 1955; Girardi et al., 1956). It was hoped that in the lung cell system t’he exact multiplicity of infection could be determined by relating the cell count to the number of EIDsO adsorbed from a given inoculum. This was not realized. While the cell counts and EID,” and HA titers are somewhat limited in their accuracy t)he errors so introduced would probably not be too serious. Other considerations, however, rendered such calculations unwarranted. (a) Trypsinized suspensions always revealed a few clumps (about 2 among 100 individual c+rlls) which were estimat,ed t,o cont,ain between 5 and 10 (*ells.Attempts t,o disperse such clumps by Verscne, although effective, tended t,o reduce HA production and this approach was, t,herefore, abandoned. (1)) The number of virus part,irles c*onstituting 1

INFLUENZA

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EID6” is not exactly known. While one infectious particle may set up infection in eggs more than one has to be injected to achieve this result. Indeed, an infectious unit may contain up to 10 virus particles, some of which may, however, be noninfective (Friedewald and Pickels, 1944; Donald and Isaacs, 1954). (c) As has been discussed above, the suspensions contained cells differing in size and possibly response to infection so that they vary presumably also in their adsorptive capacity. Because of these uncertainties it is not possible to determine under what conditions noninfectious hemagglutinins were formed. If one restricts this analysis to the number of EIDbo added per cell it is seen that with an inoculum of more than 1 EIDso per cell noninfectious HA became detectable in the progeny and the amounts produced increased as the dose of seed virus was raised to about 30 EID,, per cell. At this stage a saturation point apparently was reached. These results are compatible in principle with results obtained in chick embryos, which also showed t’hat, multiple infection of the entodermal cells of the allantois is a prerequisite for noninfectious HA production (von Magnus, 1951, 1954; Henle, 1953; Horsfall, 1954, 1955; Finter et al., 1955). It is evident that the chick embryo lung system, as employed, presents difficulties in quantitative interpretation of interactions with influenza virus. However, since some of the cells available in the suspensions appear to be fully susceptible, efforts are being made to develop a line which can be kept in continuous culture and t’o select from it, if necessary, the appropriate cells.

U~JRNET, ments Quant. CAIRSS,

F. M., and LIND, 1’. I<. using the deembryonated Riol. 18, 21-24. H. J. F. (1955). Multiplicity

(1953).

Influenza,

egg technique. reactivation

virus

(‘oltl

rcconhination:

Spring

of influenza

Harbor virus.

experi-

Symposia J. Zn~~~~cno/.

75, 326-329. I~EINHARDT, F., and HENLE, G. (1957). Studies on the viral spect,ra of tissue cult,ure lines of human cells. J. Zmnzunol. in press. DONALD, H. B. and ISAACS, A. (1954). C ounts of influenza virus particles. J. Gen. Microbial. 10, 457-462. FAZEKAS DE ST. GROTH, S., and CAIRNS, H. J. F. (1952). Quantitative aspects of virus mult.iplication. IV. Definition of constants and general discussion. 1. Znw~unol. 69, 183-181. F.~ZEK.~S UE ST. GROTH, S., and GRAHAM, 11. M. (1954). The product,ion of incomplete virus particles among influenza strains. IXxperiments in eggs. Rrit. J. Exptl. Pathol. 36, 6&74.

124

LEDINKO,

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AND

HENLE

N. B., Lru, 0. C., LIEBERMAN, M., and HENLE, W. (1954). Studies on host-virus interactions in the chick embryo-influenza virus system. VIII. An experimental analysis of various deembryonation techniques. J. Exptl. Med. 100, 33-70. FIXTER, N. B., LIU, 0. C., and HENLE, W. (1955). Studies on host-virus interactions in the chick embryo-influenza virus system. X. An experimental analysis of the von Magnus phenomenon. J. Exptl. Med. 101, 461-478. Foot, J. (1955). Relation between multiplicity of exposure, adsorption, and cytopathogenic effect of poliomyelitis virus in monkey kidney tissue cultures. Virology 1, 326333. FRIEDEWALD, W. F., and PICKEI,S, E. G. (1944). Centrifugation and ultrafiltration studies on allantoic fluid preparations of influenza virus. J. Exptl. Med. 79, 301-317. GIRARDI, A. J., MCMICHAEL, H., and HENLE, W. (1956). The use of HeLa cells in suspension for the quantitative study of virus propagation. Vit&ogv 2, 53Z-544. GRANOFF, A. (1955). Plaque formation with influenza strains. Virology 1, 252~-254. HENLE, W. (1949). Studies on host-virus interactions in the chick embryo-influenza virus system. I. Adsorption and recovery of seed virus. J. Exptl. Med. 90, I-11. HESLE, W. (1953). Developmental cycles in animal viruses. Cold Spring Harbor Symposia Quant. Biol. 18, 3534. HENLE, W., and HENLE, G. (1949). Studies on host-virus interactions in the chick embryo-influenza virus system. III. Development of infectivity, hemagglutination, and complement fixation activities during the first infectious cycle. J. Exptl. Med. 90, 23-27. HER’I,E, G., GIRARDI, il. J., and HENLE, W. (1955). A non-transmissible cytopathogenic effect of influenza virus in tissue culture accompanied by formation of noninfectious hemagglutinins. J. Exptl. Med. 101, 2541. HENLE, W., HENLE, G., and ROSENBERG, E. B. (1947). Demonstration of one-step growth curves of influenza virus through the blocking effect of irradiated virus on fm%her infection. J. Exptl. Med. 86, 423-437. HESLE, W., Lrr;, 0. C., and FINTER, N. B. (1954). Studies on host-virus interactions in the chick embryo-influenza virus system. IX. The period of liberation of virus from infected cells. J. Exptl. Med. 100, 53-70. HORSFALL, F. L. (1954). On the reproduction of influenza virus. Quantitative studies with procedures which enumerate infective and hemagglutinat,ing virus particles. J. Exptl. Med. 100, 135161. HORWALL, F. L. (1955). Reproduction of influenza viruses. Quantitative investigations with particle enumeration procedures on the dynamics of influenza A and B virus reproduction. J. Exptl. Med. 102, 441-473. KNIGHT, C. A. (1944). Titrat,ion of influenza virus in chick embryo. J. Exptl. Med. 79, 487-495. LEDINKO, N. (1955). Production of plaques with influenza viruses. Nature 176, 999-1001. PEARSON, H. E., and I+:NI>ERS, J. F. (1941). Cultivation of influenza A virus in roller tubes. P,oc,. Sot. Erpti. Riol. Med. 46, 14c-143. FINTER,

INFLUENZA

VIRUS

IN

CHICK

EMBRYO

LUNG

CELLS

125

J. D. (1948). Prevention of virus infection with enzyme of V. cholerae. I. Studies with viruses of mumps-influenza group in chick embryos. Australian

STONE,

J. Exptl. TYRRELL,

Biol.

Med.

Sci.

26, 49-64.

A. J. (1955). New tissue culture systems for influenza, Newcastle disease and vaccinia viruses. J. Immunol. 74, 293-305. VON MAGNUS, I’. (1951). Propagation of the PR8 strain of influenza A virus in chick embryos. II. The formation of “incomplete” virus following inoculation of large doses of seed virus. Acta Pathol. Microbial. Stand. 28, 278-293. VON MAGNUS, P. (1954). Incomplete forms of influenza virus. Advances in Virus Research

2, 59-79.