PRIMARY ISOLATION OF INFLUENZA VIRUSES AT 33°C

1301

and there was no evidence of adenitis. The diabetic ketosis corrected by intravenous fluids and insulin.

was

the Royal Hospital for Sick Children, His parents now reported that his Glasgow, sisters, aged 9 and 6, had developed obvious mumps on June 18. The patient was a plump baby, 1 -4 kg. more than his expected weight. He was now well hydrated. There was no glandular enlargement or any other abnormal clinical sign. The diabetes was satisfactorily controlled on 900-calorie diet and soluble

He

was

transferred

insulin.

to

June 19.

on

,

He remained constipated until June 22. Thereafter for 2 weeks he had two to five frequently loose stools each day. Bacteriological examination of the stool showed no pathogenic organism, and no virus was isolated on culture of the stool in human amnion and in rhesus monkey kidney tissue. Complement-fixation tests gave titres for both s and v mumps antigens indicating a recent mumps infection: Complement-fixatio3

Tests -

.

--

quent, there may be a more significant relation between diabetes and those cases of mumps where the main site of infection is the pancreas. I wish to thank Dr. R. A. Shanks for his advice and for permission to report this case, and also Dr. C. A. Ross of the virus laboratory, Ruchill Hospital, for the virology. REFERENCES

Gundesen, E. (1927) J. infect. Dis. 41, 197. Hinden, E. (1962) Lancet, i, 1381. Lancet (1961) i, 1389. Melin, K., Ursing, B. (1958) Nord. Med. 60, 175. Rabinovitch, I. M. (1932) Canad. med. Ass. J. 26, 551.

PRIMARY ISOLATION OF INFLUENZA VIRUSES AT 33°C H. STERN Ph.D., M.B. Glasg.

K. C. TIPPETT

CONSULTANT VIROLOGIST

SENIOR TECHNOLOGIST

DEPARTMENT OF ST.

The child was discharged from hospital on July 24. His diabetes persisted, and 6 months later was moderately well controlled on 28 units of soluble insulin daily. There has been no recurrence of diarrhoea. He is thriving and there is no of suggestion any malabsorptive defect.

Discussion

The complement-fixation tests gave values unusually high for any mumps infection. One can therefore be confident that in a 10-month-old child they indicate a recent mumps infection. He was under careful observation throughout his illness, and no sialadenitis or other apparent manifestation of mumps was seen. The occurrence of diabetes mellitus and the association of a period of diarrhoea indicate the pancreas as the probable site of infection with mumps virus. The cause of diabetes mellitus, when it occurs spontaneously, is unknown, but it is generally thought to be due to some hereditary and extrapancreatic mechanism. Infections seem to play little part in the xtiology, although -because of the impairment of carbohydrate-tolerance produced by infections (Rabinovitch 1932)-the diagnosis is not uncommonly made in the course of a pyogenic infection. In a few cases, however, the onset of diabetes mellitus may be more directly related to preceding mumps infection. In reporting such a case, Hinden (1962) was able to refer to some twenty others. The smallness of this number seems oddly at variance with the observations of Melin and Ursing (1958), and especially Gundesen (1927), who showed, at a time when diabetes mellitus was fatal, that the death-rate from diabetes in young people rose to a peak after each epidemic of mumps. The explanation may be that during an epidemic of mumps there are many cases of pancreatitis without evidence of parotitis. In these circumstances the diagnosis of mumps pancreatitis will seldom be made. If it can be assumed that diabetes is most likely to ensue in these cases, where the main weight of the mumps infection falls on the pancreas, then a significant relation between diabetes and preceding mumps infection may be passing unnoticed.

GEORGE’S

F.I.M.L.T.

MICROBIOLOGY,

HOSPITAL MEDICAL

SCHOOL, LONDON, S.W.1

EGGS are generally regarded as more sensitive than tissue culture for the primary isolation of influenza type-A viruses, yielding isolation-rates of 30-60% as compared with 2-15% in tissue culture (Mogabgab et al. 1958, Expert Committee on Respiratory Diseases 1959, Kalter et al. 1959). In this laboratory for some time it has been routine practice to culture all material from respiratory infections in tissue cultures not only at 37°C but also in a roller drum at 33 °C for the isolation of common-cold viruses. During February, 1963, there was an outbreak of influenza at St. George’s Hospital which affected the nursing staff predominantly, and the results of virus isolations suggested that tissue culture at 33°C is as sensitive as egg inoculation for the isolation of type-A influenza virus. Procedure Throat washings from five cases of influenza were inoculated, shortly after receipt, into 2 tubes each of primary cynomolgus-

monkey kidney, diploid human-embryonic lung, primary human-amnion, and HeLa-tissue cultures, which were then incubated at 37°C in a stationary position, and also into 2 tubes each of the monkey-kidney and diploid-cell cultures which were incubated at 33°C in a roller drum. The monkey kidney and diploid cells were maintained on medium 199 adjusted with sodium bicarbonate to a pH of 7-0-7-2. The same specimens, preserved by freezing at -70°C, were subsequently cultured in the amniotic sacs of 11-day embryonated eggs for 5 days at both 37°C and 33°C, and the amniotic fluids were then tested for hxmagglutinins with human group-0 cells. Virus was isolated from four of the five specimens both in tissue culture and in eggs at 33°C (table I). Isolations were made only in monkey-kidney-tissue culture, none of the other tissue cultures yielding virus. The cytopathic effects, which appeared after 4-5 days, consisted of scattered rounded granular cells, and these extended over a further 2-3 days to give an almost complete destruction of the cell sheet. The viruses were identified as Asian in type by neutralisation tests. In eggs at TABLE I-ISOLATION OF INFLUENZA VIRUSES IN TISSUE CULTURE AND EGGS AT 330c AND 370c

Summary A

of diabetes mellitus in a child aged 10 months is reported. Probably the onset was immediately preceded by mumps pancreatitis without involvement of the case

salivary glands. It is suggested that, although the relation between diabetes mellitus and obvious clinical mumps is infre-

c.p.E.cytopathic effect in monkey-kidney-tissue culture. Had. =ha:madsorption with guineapig red cells. H.A. =agglutination of human red cells by first-passage amniotic fluid.

1302

Preliminary

TABLE II—TITRBS OF PROTOTYPE INFLUENZA-A VIRUSES IN MONKEYKIDNEY-TISSUE CULTURE AFTER 4 DAYS AT 330C AND 370c

Communication

THE ORIGIN OF MITOTIC CELLS IN NORMAL THYMUS GRAFTS AFTER isologous grafting of normal thymus tissue in mice, there is massive death of cortical lymphoid tissue but apparent survival of reticular and medullary epithelial T.C.D. 60 per ml. elements of the graft. Several days later repopulation of the cortical regions of the thymus grafts by proliferating 33°C the amniotic fluids gave strong hxmagglutination on cells begins, and the grafts ultimately regain the primary passage. At 37°C, however, no haemagglutinin-positive lymphoid structure of thymus tissue. In such grafts characteristic fluids were obtained, but further passages of these eggs were of the volume of the graft is lymphoid tissue, over 90% not attempted. Also at 37°C in tissue culture no’ cytopathic the and vast majority of the dividing cells in thymus grafts effects were observed even after 10 days, though three speciare mens showed much haemadsorption of guineapig red cells on lymphocytes, mitoses being extremely rare in other the monkey-kidney-cell sheets. Subcultures of the isolates thymic-cell types. obtained at 33°C in tissue culture were carried out at both 33°C Previous findings 12 have suggested that the growth and 37°C. At 33°C cytopathic effects appeared within 2 days in behaviour of isologous thymus grafts in normal mice is subculture with all four isolates, and they progressed rapidly to largely controlled by age-conditioned genetically-detercomplete destruction of cell sheets; whereas at 37°C only two of mined stimuli arising from within the grafts themselves. the isolates caused lesions and these took 8 days to develop. Thus thymus grafts attain a characteristic size which is They consisted of scattered rounded cells and involved less than determined by the strain and age of the thymus at grafting, a quarter of the sheet; they did not progress but they were shed whilst parental thymus grafts in FI hybrid mice maintain after a few days to leave apparently normal sheets. All four the at hxmadsorbed The two subcultures 37°C, however, proliferative pattern of the parental strain. strongly. subcultures at 37°C which hamiadsorbed but failed to cause In analysing the factors which regulate the growth of cytopathic effects were again subcultured at 33°C and 37°C. thymus-graft tissue, it is of prime importance to determine Once again a progressive cytopathic effect occurred at 33°C, but the origin of the lymphoid cells repopulating the graft. failed to do so at 37°C. Are these cells descendants of cells surviving from the To determine whether this effect was peculiar for the strain original tissue grafted, or are they derived from host of virus isolated or whether it held for other type-A influenza or other host cells which have migrated to lymphocytes strains as well, a number of egg-adapted prototype strains were the grafts ? Miller3 has answered this question by using examined (table 11). The influence of temperature on cytothe T6-chromosome marker4 to distinguish host from pathogenicity and the greater sensitivity of monkey-kidney donor cells. He found that when AKR thymuses were tissue-culture cells at the lower temperature were confirmed. The table shows the results of titration of the viruses at 33°C grafted to normal (AKR x T6)FI mice, the grafts were and 37°C, as measured by cytopathic effects and by hxminitially repopulated predominately by dividing cells of adsorption, after 4 days’ incubation. At 33°C cytopathic effects graft origin; but that, from 2 to 6 weeks after grafting, an appeared within 2-3 days with all four strains, and they rapidly increasing proportion of cells of host origin were present progressed to involve most of the sheet. At 37°C, on the other in the grafts. In subsequent work on thymus grafts in hand, although cytopathic effects appeared after 2-4 days with neonatally thymectomised hosts, Miller reported5 a much the various dilutions, not more than a quarter of the cell sheet higher incidence of cells of host origin in the grafts when was affected and the rounded cells were shed within a day or these were examined 6 weeks after grafting. Dalmasso6 two, leaving apparently normal sheets. Haemadsorption was shown, after 4 days, only in tubes with cytopathic effects, but it has also studied the origin of the lymphoid cells in thymus was considerable even in those tubes at 37°C with very slight grafts in neonatally thymectomised hosts. He used the induction of runt disease by the injection of suspensions lesions. The final titres were regularly tenfold higher at 33°C. Similar results were obtained whether cynomolgus or rhesus of thymus-graft cells as a method of cell identification, and monkey-kidney cells were used. he too concluded that at 6 weeks the lymphoid cells of the Conclusions thymus grafts were host in origin. Conflicting data have While the number of isolations reported here is small, been reported on the origin of neoplastic lymphoid cells in thymic lymphomata arising in thymus grafts placed in the results are sufficiently striking to indicate the practical thymectomised-irradiated hosts. 7-9 Both chromosome value of an incubation temperature of 33°C for the isolamarker and transplantation criteria have been used, and tion of influenza-A viruses. In tissue culture at 37°C three out of the five specimens were positive by hsemadsorption 1. Metcalf, D., Sparrow, N., Nakamura, K., Ishidate, M. Aust. J. exp. Biol. med. Sci. 1961, 39, 441. only, whereas at 33°C four out of five produced obvious 2. Metcalf, D. ibid. 1963, 41 (in the press). effects. The results with the strains 3. Miller, J. F. A. P. in Ciba Foundation symposium on Tumour Viruses cytopathic prototype of Murine Origin (edited by G. E. W. Wolstenholme and M. O’Connor); suggest that 33°C provides the optimum conditions for p. 262. London, 1962. 4. Carter, T. C., Lyon, M. F., Phillips, R. J. S. J. Genet. 1955, 53, 154. cytopathogenicity and sensitivity. Similarly in eggs four 5. Miller, J. F. A. P. in The Role of the Thymus in Immunology (proceedspecimens were positive on primary passage at 33°C but ings of conference held in Minneapolis in November, 1962, edited by R. A. Good). 1963 (in the press). none at 37°C, though further passages at 37°C might have 6. Dalmasso, A. P. ibid. 7. Kaplan, H. S., Hirsch, B. B., Brown, M. B. Cancer Res. 1956, 16, 434. given positive results. Preliminary studies indicate that 8. Law, L. W., Potter, M. Proc. nat. Acad. Sci. 1956, 42, 160. influenza type-B viruses are also more cytopathogenic and 9. Barnes, D. W. H., Ford, C. E., Ilbery, P. L. T., Jones, K. W., Loutit, J. F. Acta Un. int. Cancr. 1959, 15, 544. grow better at 33°C. This raises the possibility that 33°C may prove more efficient than 37°C for the isolation not only of common cold viruses (Tyrrell and Parsons 1960) Expert Committee on Respiratory Diseases (1959) Wld Hlth Org. Tech. Rep. Ser. no. 170, p. 23. and influenza viruses but also of others whose normal Kalter, S. S., Casey, H. L., Jensen, K. E., Robinson, R. Q., Gorrie, R. H. habitat is the respiratory tract. (1959) Proc. Soc. exp. Biol., N.Y. 100, 367. *

Mogabgab, Tyrrell, D.

W. J., Pelon, W., A. J., Parsons, R.

Burch, G. E., Holmes, B. (1958) ibid. 99, (1960) Lancet, i, 235.

116.