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THE CULTIVATION IN HUMAN-EMBRYO CELLS OF A VIRUS (D.C.) CAUSING COLDS IN MAN
320
THE CULTIVATION IN HUMAN-EMBRYO CELLS OF A VIRUS CAUSING COLDS IN MAN D. A.
J. TYRRELL
M.D. Sheff.
(D.C.)
M. L. BYNOE M.B. London, D.T.M. &
H.
L. HAYFLICK Pennsylvania From the Common Cold Research Unit, Salisbury, Wilts, F. E. BUCKLAND D.M. Oxon.
and the Wistar
Ph.D.
Institute, Pennsylvania, U.S.A.
IN 1953 Andrewes et al. reported the cultivation in vitro of a cold virus. They had collected nasal washings from a subject, D.C., who had a cold and inoculated these into plasma-clot roller-tube cultures of primary human embryonic lung. The cultures were maintained at a temperature of 37 °C in a medium containing bovine amniotic fluid, bovine embryo extract, and horse serum, which was then the customary procedure for cultivating poliomyelitis viruses. No cytopathic effects were noted, but the culture fluids were passed to fresh cultures at intervals of three or four days and this process was repeated till the stock of cultures produced from that embryo was exhausted. Fluids from the fourth to the tenth passage produced colds when given to volunteers. Control material from cultures not inoculated with common-cold material did not produce colds. When material from further embryos was used the experiment could not be repeated, starting either with nasal washings or with culture fluids. The pH during the period of virus cultivation was not known. We attempted to cultivate the D.C. virus in primary human-embryo kidney cells maintained at 33°C in roller-tube cultures-a technique by which cytopathic viruses (rhinoviruses) can be isolated from many colds (Tyrrell and Bynoe 1961). No cytopathic effect was detected, however, and the culture fluid produced no colds in volunteers. We report here further experiments which have led to the design of a method for cultivating the D.C. virus in tissue-culture. Materials Virus stock.-In 1955 a volunteer developed a cold after being given tissue-culture fluid from the fourth passage of D.C. material in explant cultures of primary human-embryo Her nasal washings were administered to 17 9 of these developed colds; nasal washings in broth/saline were collected from them, pooled, and stored in small volumes at -60°C to -70°C. Before we began the tissueculture experiments to be described here, we showed that these washings, diluted J /10 and given as nasal drops, produced colds in 3 of 8 volunteers. It is probable, but not absolutely certain, that the virus in this pool is the same as that in the original D.C. washing; if it is, passage in tissue-culture may have altered its capacity for growth in different cells. Tissue-cultures.-Cultures of trypsin-dispersed primary human-embryo lung and of first-passage monkey-kidney cells were prepared as described earlier (Tyrrell et al. 1960, Tyrrell and Parsons 1960). Cultures of human-embryo lung were prepared by trypsinising, at intervals of three to five days, strains of cells isolated at Salisbury and adapted to continuous propagation in a medium containing 10% inactivated calf serum in medium 199 (Hayflick and Moorhead 1961); alternatively, human diploid cell strains, isolated at the Wistar Institute, were similarly passaged in 10% calf serum and Eagle’s medium. Methods with cytopathic viruses.-The infectivity of culture fluids was measured by end-point titrations and also by microplaque assays (Parsons and Tyrrell 1961). Antibodies were measured by a plaque-reduction method (Taylor-Robinson and Tyrrell 1962). According to a standard end-point method
lung fragments. volunteers.
using 100 T.C.D’50 of virus, a serum with neutralising activity (K) of 2 would have a serum-neutralisation titre of 50. Human subjects.-Volunteers of both sexes, whose ages ranged from eighteen to fifty years, were housed at this unit as described by Andrewes (1949). They lived in pairs or in threes in strict isolation. They were allotted to experimental groups at random, and until the end of the trial neither they nor the clinical observer were told whether or not they were in a control group.
Results
In
preliminary experiments, sixteen unsuccessful
attempts were made to propagate the virus in roller-tube tissue-cultures at 33°C. In nine of these, the primary human-embryo kidney used was maintained (a) in 2% calf serum and 0-25% lactalbumin hydrolysate (L.A.H.) with 0-035% sodium bicarbonate, or (b) in medium 199, either alone, or supplemented with 0-1% glucose and 0-2% bovine plasma-albumin. The cultures were observed for five to ten days, and no cytopathic effect was detected. In four experiments human-embryo kidney was used with various forms of medium 199 and no virus interference was found (Hitchcock and Tyrrell 1960). No cytopathic effect was seen in three experiments with cultures of primary human-embryo lung, in two with monkey-kidney cultures, or in one with calf-kidney cultures,
Suggestive Evidence for Growth of Virus in Cultures of Humanembryo Kidney Cells In the first successful experiment, 0-2 ml. of the pool of washings was added to cultures of primary human-embryo TABLE I-THE EFFECT OF FLUIDS FROM CULTURES INOCULATED WITH D.C. VIRUS
kidney maintained at 33° C with 1 -5 ml. of a medium containing 2% calf serum, 0-035% sodium bicarbonate, and no antibiotics. The culture fluids were changed at four, seven, and ten days. No cyptopathic effects were detected. The fluids from similar uninoculated cultures were harvested as a control, and all were stored. The results of inoculating these fluids to volunteers are shown in table J. Of the 30 volunteers given experimental material 8 developed colds, but none of the 29 who received control materials. The results suggest that the tenth-day harvests were infectious whereas fourth-day harvests were not, As reported earlier (Tyrrell and Bynoe 1961), other attempts (three in all) to pass the virus twice in similar cultures were unsuccessful. Production of Cytopathic Effect in Human Lung Fibroblasts A number of rhinoviruses isolated in primary human kidney cells from common colds in man will also grow in diploid cell strains of human foetal fibroblast (Hayflick and TABLE II-CYTOPATHIC EFFECT IN ROLLER-TUBE CULTURES OF HUMAN DIPLOID FIBROBLASTS
321 TABLE III-EFFECT OF CULTURE CONDITIONS ON DEVELOPMENT MICROPLAQUES OF D.C. VIRUS
OF
were inoculated with 1-0 ml. of a 1/10 dilution of the pool of washings. The results are summarised in table iv. The illnesses observed were typical afebrile common colds. The incubation period was two or three days. In the course of these trials 17 volunteers were given saline and none developed colds.
volunteers
’Normal bicarbonate was 0-088 °i, producing a pH of about 7 2. High bicarbonate was 0-16",, producing a pH of about 7-6. t The cultures were of human-embryo lung fibroblasts maintained in medium 199.
Moorhead 1961, Taylor-Robinson, Hucker, and Tyrrell
1962). We therefore inoculated roller-tube cultures of a number of cell lines with 0.2 ml. portions of the pool of washings (table n). A cytopathic effect developed two to five days after inoculation in four of the eleven strains of cells tested, although all the strains tested supported the growth of H.G.P. virus adapted to tissue-culture. The cytopathic effect was indistinguishable from that produced by strains such as F.E.B. and H.G.P. the cytopathic effect was readily passed serially in cells of the same strain, and after a few passages the virus would grow in ail the other cell strains-with the exception of HEL 113. The virus also rapidly produced a cytopathic effect in four of five batches of primary human-embryo kidney cells. All the resistant cell strains tested supported the growth of other M and H strains of virus which had been adapted to other tissue-cultures (Taylor-Robinson and Tyrrell 1962). Furthermore, using one resistant strain of cells, we were able to make primary isolations of virus from 3 volunteers’ washings, 1 containing an M strain and 2 containing H strains. Some Properties of the Virus Grown in Tissue-culture The virus causing the cytopathic effect was shown to be ether-stable, and passed a ’Gradocol ’ membrane of average pore-diameter 73 met (Dimmock and Tyrrell, 1962). Undiluted tissue-culture fluid was given by the intracerebral and intraperitoneal routes to day-old suckling mice and to guineapigs. No illness was observed. Antibody against the virus was detected (K > 0-2) in 8 of 14 adult human sera tested. The virus was not neutralised by antisera against polioviruses types 1-3, ECHO viruses types 1-9 and 11-28, Coxsackie-B viruses types 1-6, Coxsackie-A viruses types 1-24 (18 and 22 omitted), or six serotypes of rhinovirus recently reported from the Salisbury laboratory (Taylor-Robinson and Tyrrell 1962). Table III shows that the growth of the virus is inhibited in cultures maintained at a raised pH, a raised temperature, or without rolling; this has also been true of all rhinoviruses tested so far (Tyrrell and Bynoe 1961).
Relation of the Virus to Disease in Volunteers Virus which had been passed three times in tissueculture was given to volunteers, whereas control volunteers were given fluid from uninoculated cultures. In the first experiment the WI-26 strain of cell was used and 4 of 8 volunteers given virus developed colds; but so did 2 of 7 given fluid from uninoculated cultures. In another experiment the virus was passed in HEL 110 and the results were very similar, the corresponding figures being 3 out of 4 and 2 out of 2virus was recovered from the nasal washings of the 3 volunteers given virus who developed a cold and also from 1 who did not. No virus was recovered from the volunteers who were hot given virus but nevertheless developed colds. We concluded that the tissue-culture virus could multiply in the respiratory tract of man. As some unidentified agent in the uninoculated culture fluids had apparently produced colds in the controls, we could not decide whether the D.C. virus had caused the colds in the other group. A further experiment was therefore done in which 10
Viruses were isolated by inoculating cultures of HEL-110 HEL-115 cells maintained in 2% calf serum, 0 25% L.A.H., and Hanks’ balanced salt solution, with 0-035% bicarbonate. All 4 volunteers who developed a cold were infected with the virus, but 3 of 6 who did not get colds showed laboratory evidence of infection with virus. Virus was recovered from specimens from 8 of 12 volunteers with colds and from 2 of 15 without. This difference is unlikely to have arisen by chance. The sera of 3 volunteers showed rising titres of antibody against the viruses passed in tissue-culture, these viruses must therefore be closely related antigenically to that present in the original nasal washing. Moreover, all the volunteers who developed colds had low levels of antibodies, while 4 of 6 who did not get colds had high levels. The geometric-mean anti-
or
TABLE IV-LABORATORY STUDIES ON VOLUNTEERS
INOCULATED WITH
POOLED NASAL WASHINGS CONTAINING D.C. VIRUS
*
0 no virus recovered not tested virus recovered All virus isolations were from nasal washings. Two specimens of fxces collected from volunteers B, c, and D after the onset of colds, but yielded virus.
were none
body levels of the two groups differ by a factor of ten or more. Apparently, therefore, the agent causing the cold is less likely to infect volunteers with a high level of antibody against the cultured virus. Circulating antibody has previously been shown to protect volunteers against colds induced by H.G.P. (SaI.;l/57M) virus (Bynoe et al. 1961). Discussion
Our results show that the cold-producing virus in the D.C. washing can be grown in vitro with a cytopathic effect. Thus a virus which we had, on numerous occasions, failed to cultivate in primary human-kidney cells can now be grown in vitro in a reproducible fashion. The main technical advance is the use of continuously cultivated diploid human fibroblasts of certain particularly sensitive strains. Once the virus was growing, it could readily be passed to other cell strains and other types of cell. Judged by its cytopathic effect, cultural requirements, and physical properties, this virus is indistinguishable from rhinoviruses isolated in human-embryo kidney cells, although it seems to be antigenically distinct from any others studied so far at this unit. But diploid human fibroblasts are not necessarily altogether superior to human-embryo kidney cells for virus isolation. Some strains of fibroblasts are probably better than others for this purpose. Other workers, using WI-26 cells, have isolated viruses like rhinoviruses from about 15% of washings collected from adults with colds (Hamparian et al. 1961, Johnson and Chanock 1962); higher isolation-rates have been obtained when human-
322
embryo kidney cells have been used (Hamre and Prock1961, Tyrrell and Bynoe 1961, Kendall et al. 1962). We have found, however, that many of the viruses which have been isolated in primary human-kidney cells can be readily isolated in human diploid fibroblasts; the latter have the advantage that many more cells can be obtained from one embryo. Hamparian et al. (1961) have given the name " coryzaviruses " to viruses which they have isolated from colds by using WI-26 cells. They calculate the frequency of these viruses by adding together the isolation-rates in WI-26 and human-embryo kidney cells (Hilleman et al. 1962); but we have shown that this is probably not correct. But it is also possible that other viruses, besides D.C., can be isolated in cultures of diploid fibroblasts but not in cultures of primary embryo kidney. We were surprised to find that colds were produced by now
media from uninoculated cultures. Media from cultures of other cells have not produced this effect. An infectious agent of some sort may have been responsible, but its nature is unknown. In the earlier experiments of Andrewes et al. (1953), the D.C. virus was probably transmitted in one set of cultures of primary human-embryo lung because the cells were of an unusually sensitive strain, or the medium was unusually acid. Our tissue-culture-adapted virus apparently multiplied little, if at all, in cultures rolled at 36°C (see table ill).
Summary The D.C. strain of virus in nasal washings produced a focal and progressive cytopathic effect in cultures of certain strains of diploid human-embryo fibroblasts growing in semicontinuous culture. Virus was recovered from the nasal washings of 4 volunteers who had developed colds after intranasal inoculation of the washing. There was an antibody rise in the serum of 3 of 10 volunteers who were given the
virus. The virus
ether-stable and passed a filter of average 73 m[L. Its growth was inhibited by a temperature of 36°C, a pH of 7-6, and by failure to roll the culture tube. The virus was not neutralised by antisera against any of the known enteroviruses or rhinoviruses. We wish to thank Dr. H. G. Pereira, who prepared the original pool of nasal washings, and Miss E. Bullock, who assisted in making the clinical observations. Mrs. G. Hitchcock helped in some of the earlier tissue-culture experiments, and Mrs. P. K. Brown prepared many of the diploid cell cultures. Dr. H. E. M. Kay supplied much of the human tissue. We wish to thank all these, and also the volunteers for their willing cooperation. was
pore-diameter
ADRENAL FUNCTION IN SURGICAL PATIENTS AFTER STEROID THERAPY M.B. Birm.
NORMAN E. WINSTONE F.R.C.S., F.F.A.R.C.S.
RESEARCH ASSOCIATE *
SENIOR SURGICAL REGISTRAR
PHILLIDA A. SAMPSON
BRYAN N. BROOKE M.D. Birm., M.Chir. Cantab., F.R.C.S. READER IN SURGERY UNIVERSITY OF BIRMINGHAM
SINCE surgeons have been
aware
of the
danger
of
patients who have operation collapse during received steroid treatment (Fraser et al. 1952, Salassa et al. 1953, Slaney and Brooke 1957), it has been widely recommended that patients receiving steroids within 2 years of operation should be given steroid cover (Salassa et al. 1953, Slaney and Brooke 1957, Bayliss 1958). It has seemed reasonable to cover all such patients, thus providing steroids unnecessarily for many to safeguard the few. But there is evidence that indiscriminate steroid cover may expose such patients to an increased incidence of infection of a serious nature (Winstone and Brooke 1961). It has therefore become important to determine which patients need operative cover after steroid therapy. With this in view, the adrenal function of such cases has been tested so as to make the application of steroid " cover " selective instead of routine, and the subsequent plasma-cortisol levels during surgery and afterwards have been measured. The corticotrophin test has been used to measure adrenal function in cases of Addison’s and Cushing’s disease (Bliss et al. 1954, Eik-Nes et al. 1954,Christy et al. 1955, 1956, Holub et al. 1959); the adrenal response to surgery has been measured by estimation of free and conjugated plasma-cortisol both during operation and postoperatively by Sandberg et al. (1954), Steenburg etal. (1956), Helmreich et al. (1957), LeFemine et al. (1957), Moore (1957), and Marks et al. (1959). So far as we know, this work has been applied to the study of adrenal function in patients treated with steroids preoperatively only by Marks et al. (1959) and this correlated with the response to operation. Our study has been conducted along similar lines, but with a view to determining the need or otherwise for steroid cover during and after operation. It was necessary to consider what form of test would meet the requirements and exigencies of clinical practice with sufficient accuracy. The eosinophil response to corticotrophin (Thorn’s test), though simple in execution, or
*
in
after
Supported by
a
M.R.C. grant.
REFERENCES
Andrewes, C. H. (1949) Lancet, i, 71. Chaproniere, D. M., Gompels, A. E. H., Pereira, H. G., Roden, A. T. (1953) ibid. ii, 546. Bynoe, M. L., Hobson, D., Horner, J., Kipps, A., Schild. G. C. Tyrrell, D. A. J. (1961) ibid. i, 1194. Dimmock, N., Tyrrell, D. A. J. (1962) Unpublished. Hamparian, V. V., Ketler, A., Hilleman, M. R. (1961) Proc. Soc. exp. Biol., N.Y. 108, 444 Hamre, D., Procknow, J. (1961) Brit. med. J.. ii, 770. Hayflick, L., Moorhead, P. S. (1961) Exp. cell. Res. 25, 585. Hilleman, M. R., Hamparian, V. V., Ketler, A., Reilly, C. M., McClelland, L., Cornfeld, D., Stokes, J. (1962) J. Amer. med. Ass. 180, 445. Hitchcock, G., Tyrrell, D. A. J. (1960) Lancet, i, 237. Johnson, K., Chanock, R. M. (1962) Personal communication. Kendall, E. J. C., Bynoe, M. L., Tyrrell, D. A. J. (1962) Brit. med. J. ii, 82. Parsons, R., Tyrrell, D. A. J. (1961) Nature, Lond. 189, 640. Taylor-Robinson, D., Hucker, R., Tyrrell, D. A. J. (1962) Brit. J. exp. Path. 43, 189. — Tyrrell, D. A. J. (1962) Lancet, i, 452. Tyrrell, D. A. J., Bynoe, M. L. (1961) Brit. med. J. i, 393. Hitchcock, G., Pereira, H. G., Andrewes, C. H. (1960) Lancet, i, 235. Parsons, R. (1960) ibid. p. 239. —
—
—
—
Fig. 1-Corticotrophin tests: the shaded areas show the range of plasma-cortisol levels and the mean values for the two groups. In the steroid group, unsatisfactory responses are indicated by broken lines.
THE CULTIVATION IN HUMAN-EMBRYO CELLS OF A VIRUS CAUSING COLDS IN MAN D. A.
J. TYRRELL
M.D. Sheff.
(D.C.)
M. L. BYNOE M.B. London, D.T.M. &
H.
L. HAYFLICK Pennsylvania From the Common Cold Research Unit, Salisbury, Wilts, F. E. BUCKLAND D.M. Oxon.
and the Wistar
Ph.D.
Institute, Pennsylvania, U.S.A.
IN 1953 Andrewes et al. reported the cultivation in vitro of a cold virus. They had collected nasal washings from a subject, D.C., who had a cold and inoculated these into plasma-clot roller-tube cultures of primary human embryonic lung. The cultures were maintained at a temperature of 37 °C in a medium containing bovine amniotic fluid, bovine embryo extract, and horse serum, which was then the customary procedure for cultivating poliomyelitis viruses. No cytopathic effects were noted, but the culture fluids were passed to fresh cultures at intervals of three or four days and this process was repeated till the stock of cultures produced from that embryo was exhausted. Fluids from the fourth to the tenth passage produced colds when given to volunteers. Control material from cultures not inoculated with common-cold material did not produce colds. When material from further embryos was used the experiment could not be repeated, starting either with nasal washings or with culture fluids. The pH during the period of virus cultivation was not known. We attempted to cultivate the D.C. virus in primary human-embryo kidney cells maintained at 33°C in roller-tube cultures-a technique by which cytopathic viruses (rhinoviruses) can be isolated from many colds (Tyrrell and Bynoe 1961). No cytopathic effect was detected, however, and the culture fluid produced no colds in volunteers. We report here further experiments which have led to the design of a method for cultivating the D.C. virus in tissue-culture. Materials Virus stock.-In 1955 a volunteer developed a cold after being given tissue-culture fluid from the fourth passage of D.C. material in explant cultures of primary human-embryo Her nasal washings were administered to 17 9 of these developed colds; nasal washings in broth/saline were collected from them, pooled, and stored in small volumes at -60°C to -70°C. Before we began the tissueculture experiments to be described here, we showed that these washings, diluted J /10 and given as nasal drops, produced colds in 3 of 8 volunteers. It is probable, but not absolutely certain, that the virus in this pool is the same as that in the original D.C. washing; if it is, passage in tissue-culture may have altered its capacity for growth in different cells. Tissue-cultures.-Cultures of trypsin-dispersed primary human-embryo lung and of first-passage monkey-kidney cells were prepared as described earlier (Tyrrell et al. 1960, Tyrrell and Parsons 1960). Cultures of human-embryo lung were prepared by trypsinising, at intervals of three to five days, strains of cells isolated at Salisbury and adapted to continuous propagation in a medium containing 10% inactivated calf serum in medium 199 (Hayflick and Moorhead 1961); alternatively, human diploid cell strains, isolated at the Wistar Institute, were similarly passaged in 10% calf serum and Eagle’s medium. Methods with cytopathic viruses.-The infectivity of culture fluids was measured by end-point titrations and also by microplaque assays (Parsons and Tyrrell 1961). Antibodies were measured by a plaque-reduction method (Taylor-Robinson and Tyrrell 1962). According to a standard end-point method
lung fragments. volunteers.
using 100 T.C.D’50 of virus, a serum with neutralising activity (K) of 2 would have a serum-neutralisation titre of 50. Human subjects.-Volunteers of both sexes, whose ages ranged from eighteen to fifty years, were housed at this unit as described by Andrewes (1949). They lived in pairs or in threes in strict isolation. They were allotted to experimental groups at random, and until the end of the trial neither they nor the clinical observer were told whether or not they were in a control group.
Results
In
preliminary experiments, sixteen unsuccessful
attempts were made to propagate the virus in roller-tube tissue-cultures at 33°C. In nine of these, the primary human-embryo kidney used was maintained (a) in 2% calf serum and 0-25% lactalbumin hydrolysate (L.A.H.) with 0-035% sodium bicarbonate, or (b) in medium 199, either alone, or supplemented with 0-1% glucose and 0-2% bovine plasma-albumin. The cultures were observed for five to ten days, and no cytopathic effect was detected. In four experiments human-embryo kidney was used with various forms of medium 199 and no virus interference was found (Hitchcock and Tyrrell 1960). No cytopathic effect was seen in three experiments with cultures of primary human-embryo lung, in two with monkey-kidney cultures, or in one with calf-kidney cultures,
Suggestive Evidence for Growth of Virus in Cultures of Humanembryo Kidney Cells In the first successful experiment, 0-2 ml. of the pool of washings was added to cultures of primary human-embryo TABLE I-THE EFFECT OF FLUIDS FROM CULTURES INOCULATED WITH D.C. VIRUS
kidney maintained at 33° C with 1 -5 ml. of a medium containing 2% calf serum, 0-035% sodium bicarbonate, and no antibiotics. The culture fluids were changed at four, seven, and ten days. No cyptopathic effects were detected. The fluids from similar uninoculated cultures were harvested as a control, and all were stored. The results of inoculating these fluids to volunteers are shown in table J. Of the 30 volunteers given experimental material 8 developed colds, but none of the 29 who received control materials. The results suggest that the tenth-day harvests were infectious whereas fourth-day harvests were not, As reported earlier (Tyrrell and Bynoe 1961), other attempts (three in all) to pass the virus twice in similar cultures were unsuccessful. Production of Cytopathic Effect in Human Lung Fibroblasts A number of rhinoviruses isolated in primary human kidney cells from common colds in man will also grow in diploid cell strains of human foetal fibroblast (Hayflick and TABLE II-CYTOPATHIC EFFECT IN ROLLER-TUBE CULTURES OF HUMAN DIPLOID FIBROBLASTS
321 TABLE III-EFFECT OF CULTURE CONDITIONS ON DEVELOPMENT MICROPLAQUES OF D.C. VIRUS
OF
were inoculated with 1-0 ml. of a 1/10 dilution of the pool of washings. The results are summarised in table iv. The illnesses observed were typical afebrile common colds. The incubation period was two or three days. In the course of these trials 17 volunteers were given saline and none developed colds.
volunteers
’Normal bicarbonate was 0-088 °i, producing a pH of about 7 2. High bicarbonate was 0-16",, producing a pH of about 7-6. t The cultures were of human-embryo lung fibroblasts maintained in medium 199.
Moorhead 1961, Taylor-Robinson, Hucker, and Tyrrell
1962). We therefore inoculated roller-tube cultures of a number of cell lines with 0.2 ml. portions of the pool of washings (table n). A cytopathic effect developed two to five days after inoculation in four of the eleven strains of cells tested, although all the strains tested supported the growth of H.G.P. virus adapted to tissue-culture. The cytopathic effect was indistinguishable from that produced by strains such as F.E.B. and H.G.P. the cytopathic effect was readily passed serially in cells of the same strain, and after a few passages the virus would grow in ail the other cell strains-with the exception of HEL 113. The virus also rapidly produced a cytopathic effect in four of five batches of primary human-embryo kidney cells. All the resistant cell strains tested supported the growth of other M and H strains of virus which had been adapted to other tissue-cultures (Taylor-Robinson and Tyrrell 1962). Furthermore, using one resistant strain of cells, we were able to make primary isolations of virus from 3 volunteers’ washings, 1 containing an M strain and 2 containing H strains. Some Properties of the Virus Grown in Tissue-culture The virus causing the cytopathic effect was shown to be ether-stable, and passed a ’Gradocol ’ membrane of average pore-diameter 73 met (Dimmock and Tyrrell, 1962). Undiluted tissue-culture fluid was given by the intracerebral and intraperitoneal routes to day-old suckling mice and to guineapigs. No illness was observed. Antibody against the virus was detected (K > 0-2) in 8 of 14 adult human sera tested. The virus was not neutralised by antisera against polioviruses types 1-3, ECHO viruses types 1-9 and 11-28, Coxsackie-B viruses types 1-6, Coxsackie-A viruses types 1-24 (18 and 22 omitted), or six serotypes of rhinovirus recently reported from the Salisbury laboratory (Taylor-Robinson and Tyrrell 1962). Table III shows that the growth of the virus is inhibited in cultures maintained at a raised pH, a raised temperature, or without rolling; this has also been true of all rhinoviruses tested so far (Tyrrell and Bynoe 1961).
Relation of the Virus to Disease in Volunteers Virus which had been passed three times in tissueculture was given to volunteers, whereas control volunteers were given fluid from uninoculated cultures. In the first experiment the WI-26 strain of cell was used and 4 of 8 volunteers given virus developed colds; but so did 2 of 7 given fluid from uninoculated cultures. In another experiment the virus was passed in HEL 110 and the results were very similar, the corresponding figures being 3 out of 4 and 2 out of 2virus was recovered from the nasal washings of the 3 volunteers given virus who developed a cold and also from 1 who did not. No virus was recovered from the volunteers who were hot given virus but nevertheless developed colds. We concluded that the tissue-culture virus could multiply in the respiratory tract of man. As some unidentified agent in the uninoculated culture fluids had apparently produced colds in the controls, we could not decide whether the D.C. virus had caused the colds in the other group. A further experiment was therefore done in which 10
Viruses were isolated by inoculating cultures of HEL-110 HEL-115 cells maintained in 2% calf serum, 0 25% L.A.H., and Hanks’ balanced salt solution, with 0-035% bicarbonate. All 4 volunteers who developed a cold were infected with the virus, but 3 of 6 who did not get colds showed laboratory evidence of infection with virus. Virus was recovered from specimens from 8 of 12 volunteers with colds and from 2 of 15 without. This difference is unlikely to have arisen by chance. The sera of 3 volunteers showed rising titres of antibody against the viruses passed in tissue-culture, these viruses must therefore be closely related antigenically to that present in the original nasal washing. Moreover, all the volunteers who developed colds had low levels of antibodies, while 4 of 6 who did not get colds had high levels. The geometric-mean anti-
or
TABLE IV-LABORATORY STUDIES ON VOLUNTEERS
INOCULATED WITH
POOLED NASAL WASHINGS CONTAINING D.C. VIRUS
*
0 no virus recovered not tested virus recovered All virus isolations were from nasal washings. Two specimens of fxces collected from volunteers B, c, and D after the onset of colds, but yielded virus.
were none
body levels of the two groups differ by a factor of ten or more. Apparently, therefore, the agent causing the cold is less likely to infect volunteers with a high level of antibody against the cultured virus. Circulating antibody has previously been shown to protect volunteers against colds induced by H.G.P. (SaI.;l/57M) virus (Bynoe et al. 1961). Discussion
Our results show that the cold-producing virus in the D.C. washing can be grown in vitro with a cytopathic effect. Thus a virus which we had, on numerous occasions, failed to cultivate in primary human-kidney cells can now be grown in vitro in a reproducible fashion. The main technical advance is the use of continuously cultivated diploid human fibroblasts of certain particularly sensitive strains. Once the virus was growing, it could readily be passed to other cell strains and other types of cell. Judged by its cytopathic effect, cultural requirements, and physical properties, this virus is indistinguishable from rhinoviruses isolated in human-embryo kidney cells, although it seems to be antigenically distinct from any others studied so far at this unit. But diploid human fibroblasts are not necessarily altogether superior to human-embryo kidney cells for virus isolation. Some strains of fibroblasts are probably better than others for this purpose. Other workers, using WI-26 cells, have isolated viruses like rhinoviruses from about 15% of washings collected from adults with colds (Hamparian et al. 1961, Johnson and Chanock 1962); higher isolation-rates have been obtained when human-
322
embryo kidney cells have been used (Hamre and Prock1961, Tyrrell and Bynoe 1961, Kendall et al. 1962). We have found, however, that many of the viruses which have been isolated in primary human-kidney cells can be readily isolated in human diploid fibroblasts; the latter have the advantage that many more cells can be obtained from one embryo. Hamparian et al. (1961) have given the name " coryzaviruses " to viruses which they have isolated from colds by using WI-26 cells. They calculate the frequency of these viruses by adding together the isolation-rates in WI-26 and human-embryo kidney cells (Hilleman et al. 1962); but we have shown that this is probably not correct. But it is also possible that other viruses, besides D.C., can be isolated in cultures of diploid fibroblasts but not in cultures of primary embryo kidney. We were surprised to find that colds were produced by now
media from uninoculated cultures. Media from cultures of other cells have not produced this effect. An infectious agent of some sort may have been responsible, but its nature is unknown. In the earlier experiments of Andrewes et al. (1953), the D.C. virus was probably transmitted in one set of cultures of primary human-embryo lung because the cells were of an unusually sensitive strain, or the medium was unusually acid. Our tissue-culture-adapted virus apparently multiplied little, if at all, in cultures rolled at 36°C (see table ill).
Summary The D.C. strain of virus in nasal washings produced a focal and progressive cytopathic effect in cultures of certain strains of diploid human-embryo fibroblasts growing in semicontinuous culture. Virus was recovered from the nasal washings of 4 volunteers who had developed colds after intranasal inoculation of the washing. There was an antibody rise in the serum of 3 of 10 volunteers who were given the
virus. The virus
ether-stable and passed a filter of average 73 m[L. Its growth was inhibited by a temperature of 36°C, a pH of 7-6, and by failure to roll the culture tube. The virus was not neutralised by antisera against any of the known enteroviruses or rhinoviruses. We wish to thank Dr. H. G. Pereira, who prepared the original pool of nasal washings, and Miss E. Bullock, who assisted in making the clinical observations. Mrs. G. Hitchcock helped in some of the earlier tissue-culture experiments, and Mrs. P. K. Brown prepared many of the diploid cell cultures. Dr. H. E. M. Kay supplied much of the human tissue. We wish to thank all these, and also the volunteers for their willing cooperation. was
pore-diameter
ADRENAL FUNCTION IN SURGICAL PATIENTS AFTER STEROID THERAPY M.B. Birm.
NORMAN E. WINSTONE F.R.C.S., F.F.A.R.C.S.
RESEARCH ASSOCIATE *
SENIOR SURGICAL REGISTRAR
PHILLIDA A. SAMPSON
BRYAN N. BROOKE M.D. Birm., M.Chir. Cantab., F.R.C.S. READER IN SURGERY UNIVERSITY OF BIRMINGHAM
SINCE surgeons have been
aware
of the
danger
of
patients who have operation collapse during received steroid treatment (Fraser et al. 1952, Salassa et al. 1953, Slaney and Brooke 1957), it has been widely recommended that patients receiving steroids within 2 years of operation should be given steroid cover (Salassa et al. 1953, Slaney and Brooke 1957, Bayliss 1958). It has seemed reasonable to cover all such patients, thus providing steroids unnecessarily for many to safeguard the few. But there is evidence that indiscriminate steroid cover may expose such patients to an increased incidence of infection of a serious nature (Winstone and Brooke 1961). It has therefore become important to determine which patients need operative cover after steroid therapy. With this in view, the adrenal function of such cases has been tested so as to make the application of steroid " cover " selective instead of routine, and the subsequent plasma-cortisol levels during surgery and afterwards have been measured. The corticotrophin test has been used to measure adrenal function in cases of Addison’s and Cushing’s disease (Bliss et al. 1954, Eik-Nes et al. 1954,Christy et al. 1955, 1956, Holub et al. 1959); the adrenal response to surgery has been measured by estimation of free and conjugated plasma-cortisol both during operation and postoperatively by Sandberg et al. (1954), Steenburg etal. (1956), Helmreich et al. (1957), LeFemine et al. (1957), Moore (1957), and Marks et al. (1959). So far as we know, this work has been applied to the study of adrenal function in patients treated with steroids preoperatively only by Marks et al. (1959) and this correlated with the response to operation. Our study has been conducted along similar lines, but with a view to determining the need or otherwise for steroid cover during and after operation. It was necessary to consider what form of test would meet the requirements and exigencies of clinical practice with sufficient accuracy. The eosinophil response to corticotrophin (Thorn’s test), though simple in execution, or
*
in
after
Supported by
a
M.R.C. grant.
REFERENCES
Andrewes, C. H. (1949) Lancet, i, 71. Chaproniere, D. M., Gompels, A. E. H., Pereira, H. G., Roden, A. T. (1953) ibid. ii, 546. Bynoe, M. L., Hobson, D., Horner, J., Kipps, A., Schild. G. C. Tyrrell, D. A. J. (1961) ibid. i, 1194. Dimmock, N., Tyrrell, D. A. J. (1962) Unpublished. Hamparian, V. V., Ketler, A., Hilleman, M. R. (1961) Proc. Soc. exp. Biol., N.Y. 108, 444 Hamre, D., Procknow, J. (1961) Brit. med. J.. ii, 770. Hayflick, L., Moorhead, P. S. (1961) Exp. cell. Res. 25, 585. Hilleman, M. R., Hamparian, V. V., Ketler, A., Reilly, C. M., McClelland, L., Cornfeld, D., Stokes, J. (1962) J. Amer. med. Ass. 180, 445. Hitchcock, G., Tyrrell, D. A. J. (1960) Lancet, i, 237. Johnson, K., Chanock, R. M. (1962) Personal communication. Kendall, E. J. C., Bynoe, M. L., Tyrrell, D. A. J. (1962) Brit. med. J. ii, 82. Parsons, R., Tyrrell, D. A. J. (1961) Nature, Lond. 189, 640. Taylor-Robinson, D., Hucker, R., Tyrrell, D. A. J. (1962) Brit. J. exp. Path. 43, 189. — Tyrrell, D. A. J. (1962) Lancet, i, 452. Tyrrell, D. A. J., Bynoe, M. L. (1961) Brit. med. J. i, 393. Hitchcock, G., Pereira, H. G., Andrewes, C. H. (1960) Lancet, i, 235. Parsons, R. (1960) ibid. p. 239. —
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Fig. 1-Corticotrophin tests: the shaded areas show the range of plasma-cortisol levels and the mean values for the two groups. In the steroid group, unsatisfactory responses are indicated by broken lines.