STUDIES WITH HUMAN INFLUENZA VIRUS CULTIVATED IN ARTIFICIAL MEDIUM

Published Online: 1 June, 1936 Supp Inf: http://di.rg/1.184/jem.63.6.83 Dwnladed frm jem.rupress.rg n August 13, 218 STUDIES WITH HUMAN INFLUENZA VIRUS CULTIVATED IN ARTIFICIAL MEDIUM BY T. P. MAGILL, M.D., Am) THOMAS FRANCIS, Jl~., M.D. (Frm the Hspital f The Rckefeller Institute fr Medical Research) (Received fr publicatin, January 14, 1936) A previus reprt (1) has recrded the successful cultivatin f a strain f human influenza virus (P.R.8) (2) in an artificial medium cmpsed f Tyrde's slutin and minced chick embry. The virus cultivated under these cnditins was fund t retain its capacity t infect mice and ferrets, and it was maintained at abut the same cncentratn as in the lungs f infected mice frm which it had been derived. Since the preliminary reprt the P.R.8 strain f human influenza virus has been cntinuusly transferred at 2 day intervals until it is nw in the 7th generatin. In additin, under similar cnditins, a 2nd strain f human influenza virus (Philadelphia) (3) has been cultivated thrugh 45 successive transfers in the artificial medium. Still mre recently the virus f swine influenza (4), btained frm the lungs f infected mice) has als been transferred t tissue culture. Burnet (5) and Smith (6) have reprted the successful cultivatin f strains f human influenza virias upn the chriallantic membranes f develping eggs. The latter has als cnfirmed ur results in cultivating the virus in a fluid tissue medium. Certain bservatins have been made regarding the cnditins affecting multiplicatin and the time f survival f the virus. Furthermre, studies have been made f the immunlgical prperties f culture virus as cmpared with the same strain f virus maintained slely by passage thrugh susceptible animals. The present paper cmprises primarily the results f these latter investigatins. Methds and Materials Culture Mediura.--The medium emplyed is that devised by Li and Rivers (7) based upn the earlier prcedure f Maitland and Maitland (8). Chick em- x Thrugh the curtesy f Dr. Shpe. 83

84 HITMAN INFLUENZA VIRUS IN ARTIFICIAL MEDIUM brys after 1 t 14 days' incubatin are remved aseptically frm the egg, the eyes are taken ut, and the remainder f the embry is finely minced in 2 t 4 cc. f Tyrde's slutin, depending upn the size f the embry. After the preliminary perid, embrys f 12 t 13 days were unifrmly used. T 4.5 cc. f Tyrde's slutin in a Rivers flask r in a 5 cc. Erlenmeyer flask, are added 4 drps (apprximately.25 cc.) f the suspensin f embrynic tissue. T the medium is then added.5 cc. f the virus-cntaining material. The flasks are stppered with firm plugs f cttn bund in gauze. After 2 days' incubatin at 37 C., transfers f.5 cc. f the culture are made t flasks cntaining 4.5 cc. f freshly prepared medium. Fr rutine purpses the transfers have subsequently been made at 2 day intervals. In this manner, the P.R.8 strain has been actively maintained in culture fr ver 4 mnths and the Philadelphia strain fr a smewhat shrter perid. A cntrl culture nt cntaining virus has als been transferred as rutine. Methd f Titratin.--The presence f active virus in the culture fluid has been demnstrated by the instillatin f the material int the nstrils f white mice lightly anesthetized with ether. The active agent induces in these animals grss pulmnary cnslidatin which varies in extent with the cncentratin f the virus. Mice receiving.5 cc. f culture f the usual titer die r are mribund in 4 t 6 days with cmplete invlvement f the lungs. All mice surviving n the 6th day are sacrificed and their lungs examined fr grss lesins. With cultures f lwer titer nly slight lesins may be prduced in this time. Fr purpses f titratin the limit f infectiusness has been taken t be the highest dilutin f culture which prduces visible areas f invlvement in the lungs f inculated mice. It is realized, hwever, that this arbitrary limit may nt be entirely accurate, fr it has been pssible t demnstrate, by passage t nrmal mice, the presence f virus in the lungs f mice in which at the end f 6 days visible lesins were nt bserved. Nevertheless, fr practical purpses, the end-pint as measured by the abve methd has been generally emplyed. Multiplicatin and Survival f Virus under Different Cultural Cnditins Under the standard cnditins adpted, the greatest cncentratin f virus in the artificial medium is usually attained in 36 t 48 hurs. In 72 hurs a decrease in the amunt f virus has begun, and after 5 t 6 days it is difficult t demnstrate active virus in the culture fluid. Nevertheless, in cultures remved frm the incubatr after 48 hurs' incubatin and placed in the refrigeratr at +4 C., the virus has been fund t retain practically full infectiusness fr as lng as 23 days. Furthermre, cultures made with the same medium in rdinary test tubes plugged with a tight rubber stpper and incubated at 37 C. have been fund t cntain active virus as lng as 18 days. At this

T. P. MAGILL AND THOMAS FRANCIS, JR. 85 time lesins were nt bserved in mice inculated with the material, but when these cultures were transplanted t freshmedium in tightly stppered test tubes and transferred at 4 day intervals, the virus regained its full ptency and was satisfactrily maintained by this prcedure. Hwever, if vaseline seals are placed ver the culture medium s as t apprach anaerbic cnditins, the virus des nt multiply, and the culture fluid after 48 hurs is nt infectius fr mice. The effect f variatins in the amunt f tissue used in the culture medium has nt been fully investigated. As previusly recgnized, this factr plays a definite r61e in the prblem f virus cultivatin. A very small number f living cells apparently des nt supprt multiplicatin f the virus, while t great a quantity f tissue is als detrimental. In the present study embrys f 12 t 13 days have appeared t be the mst satisfactry, pssibly because the embry cntains at this time a greater amunt f serum which may serve t prtect the virus. Maintenance f Virulence f Human Influenza Virus in Tissue Culture The virulence f the culture virus, r the retentin f its capacity t prduce pulmnary lesins in susceptible mice, has been measured at frequent intervals. In mst instances, mice receiving the undiluted culture fluid succumb in 6 days r less and exhibit extensive invlvement f the lungs. Titratins f the virus cncentratin f the standard P.R.8 cultures made at different times are presented in Table I. It can be seen that a cmparatively cnstant fiter f between 1:1 and 1:1, has been maintained. Ferrets, as well as mice, have been successfully inculated with virus f the 6th, 37th, and 54th transfers f the P.R.8 strain. In each case the ferret respnded with fever, and in thse instances in which autpsies were dne invlvement f the lungs f the ferret was bserved. Furthermre, the serum f ferrets recvering frm infectin with the culture virus was fund t cntain a high cncentratin f antibdies effective against the regular muse passage virus and the animals were fund t be actively immune t reinfectin when tested with ferret passage virus.

86 HUMAN INFLUENZA VIRUS IN ARTIFICIAL MEDIU-~ Immunizatin Experiments with Culture Virus It has been reprted previusly that mice inculated subcutaneusly r intraperitneally with human influenza virus (9, 1), althugh shwing n evidence f experimental disease, develp an immunity TABLE I Titratins f P.R. 8 Culture Virus at Intervals during the Curse f Cultivatin Transfer Muse N. 1-1 1-2 1-~ Dilutin f virus 1-~ 1-~ I~ 6th * * ± + + + + ± ~- loth + + ± + 19th + + + -4- -4- + + 31st * 4-4-+ +± 4-4- -4-39th * +± 4-4-4-± 4-4-4-4-+ 42nd *+4-4-4-4-4-4- 4-+ 4-+ 4-4- ± 55~ *4-+4-4- * *4-4- + 4-4- 4- + 4-+ -- n grss pulmnary invlvement. t 4-4- 4-4- = prgressive degrees f pulmnary invlvement. * = muse died. which is effective against the virus inculated intranasally. It was f interest, therefre, t determine whether the virus prpagating in tissue culture utside the animal bdy was still capable f exerting this effect.

T. P. MAGILL AND THOMAS FRANCIS, JR. 87 Experiment/.--T each f 15 mice was administered subcutaneusly.3 cc. f P.R.8 virus f the 3rd culture transfer. 9 days and 21 days later,.3 cc. prtins f the fluid f the 7th and 13th generatins respectively were given intraperitneally. 14 mice f the same stck were kept as cntrls, receiving n inculatins. 8 days after the last injectin bth vaccinated and cntrl mice were given.3 cc. f a i per cent suspensin f P.R.8 muse passage virus intranasally. By the 1th day 11 f the 14 cntrl mice had died with extensive pulmnary invlvement; the 3 surviving cntrl mice were killed and marked pulmnary lesins were exhibited. The vaccinated mice had appeared perfectly well, and in the 5 killed n the 1th day n pulmnary lesins were bserved. Experiment 2.--Three grups f 1 mice each were used. One grup received, at 1 day intervals,.2 cc.,.3 cc.,.3 cc., f the 4th, 45th and 49th generatins, respectively, f the P.R.8 culture virus subcutaneusly; thse f the secnd grup received equal amunts f the same active virus cultures intraperitneauy at the same intervals. Animals f the third, r cntrl, grup, were given subcutaneusly.2 cc. f culture medium which cntained n virus, and at 1 day intervals thereafter tw dses f.3 cc. each f similar material were given intraperitneally. 1 days after thelast vaccinatin all mice were given.3 cc. f 1 per cent suspensin f P.R.8 muse passage virus intranasally. By the 1th day after infectin, 9 f the 1 cntrl animals had died with typical pulmnary invlvement; 2 f the mice which were vaccinated subcutaneusly had died, althugh the thers f this grup appeared perfectly well; all f the mice which had been vaccinated by the intraperitneal rute remained well thrughut the perid f bservatin. The results cited, and ther experiments f a similar nature, have shwn cnclusively that mice vaccinated subcutaneusly r intraperitneally with human influenza virus transferred thrugh many generatins in tissue culture develp a staunch immunity against intranasal infectin with large dses f the same strain f virus maintained entirely by serial passages in mice. Experiments relating t the efficacy f the culture virus in the immunizatin f ferrets have shwn that, fllwing subcutaneus inculatin f the artificially cultivated agent, the animals develp an active resistance which distinctly mdifies the disease prduced by intranasal instillatin f regular ferret passage virus. Studies in prgress regarding the vaccinatin f human individuals (11) have revealed that, fllwing the subcutaneus inculatin f virus culture fluid, the human subject respnds with a develpment f antibdies capable f neutralizing the virus as demnstrated by muse prtectin tests.

88 HUMAN INFLUENZA VIRUS IN ARTIFICIAL MEDIUM Studies f the Immunlgical Characteristics f Human Influenza Virus Grwn in A rtifidal Medium The evidence heretfre btained, thrugh titratin in white mice, f the cncentratin f active agent in culture fluid has indicated that the virus multiplying in tissue culture reaches a cncentratin and maintains a virulence clsely resembling that f the virus in the lungs f infected mice. Hwever, when the capacity f certain sera t neutralize the culture virus was cmpared with the capacity f these sera t neutralize the same strain f muse passage virus, distinct TABLE Cmparisn f Neutralizing Capacity f the Same Sera Tested against Culture Virus f 4th and 15th Transfers II Serum 15th transfer (Original culture) Muse N. Culture virus (P.R. 8) 4th transfer (New culture) Muse N. Nrmal ferret 1-5... Immune " 1-14... " + + "t- Nrma rabbit 1-33... :' l + Immune " 1-29... " swine 14-44... [ Human--H.F. (acute influenza)... " H.F. (cnvalescent influ- enza)... Human--T.F. (nrmal)... 3-7 + + + +2++ + + differences were nted. In general, the neutralizing effect f the serum was enhanced when tested against culture virus, s that sera which exhibited little r n prtective capacity against muse passage strain might prtect mice cmpletely against the cultivated virus. In the earlier phases f this study it appeared that the ease with which the virus grwn in artificial medium culd be neutralized by a given serum was smewhat related t the length f time it had been remved frm animal passages. Tests were made simultaneusly with the same sera against ne culture in its 15th transfer and a culture f the same strain which had been transferred thrugh mice

T. P. MAGILL AND THOMAS FRANCIS, JR. 89 after culture and was then returned t artificial cultivatin fr 4 generatins. The results are presented in Table II. It is readily bserved that nrmal ferret and nrmal rabbit serum exerted a strnger neutralizing effect against the lder culture virus than against the mre recent culture. Furthermre, the serum f swine cnvalescent frm swine influenza virus infectin, and f tw human beings, H.F. (acute) and T.F. cmpletely prtected against the lder culture virus. When the virus frm the 15th generatin in artificial medium was passed thrugh mice fr 3 transfers and cmparative tests were again TABLE Capacity f Varius Sera t Neutralize Culture Virus (P.R. 8) befre and after Passage thrugh Mice III Serum Culture virus (2 Ist transfer) Muse N. Culture virus (15th transfer) after 3 serial muse passages Muse N. 1 2 3 2 H. F. (acute)... H. F. (cnvalescent)... S.S. "... T. F. (nrmal)... Nrmal rabbit 1-29... Immune (P.R.8) rabbit 1-29. Nrmal swine... Swine, immune t swine influ- e]~?,a... ;+2 + + + + * L * + dne, it was fund that after animal passages the virus had regained its riginal characteristics and that neutralizatin f the virus by these sera was n lnger effected (Table III). Recently, the cnditins f cultivatin have been smewhat mre cnstant than earlier in the study, and a cmparative test was made between the same strain f cultivated virus in its 54th generatin and a new culture which was nly 5 transfers (1 days) remved frm animal passage. In this instance, the lder culture virus which had exhibited little fluctuatin in its ptency fr a cnsiderable time, was less easily neutralized than previusly and was n mre susceptible t the actin f serum than the yunger culture (Table IV).

81 ItV~AN INFLUENZA VIRUS IN ARTIFICIAL MIEDILr~ These results suggest that the variatins bserved have been due entirely t alteratins f a quantitative, nt f a qualitative nature in the virus. Certainly n change in the immunlgical characteristics has been bserved which culd be interpreted as due t an alteratin in the antigenic cnstitutin f the virus. On the ther hand, since the titer f the culture virus, as measured by the custmary methd in susceptible mice, has remained at a level clsely parallel t that f muse passage virus, the differences bserved might be attributed t a qualitative change in the virulence f individual virus particles, s that mre wuld be required t prduce a lesin equal in severity t that TABLE Cmparis~ f Neutralizing Capacity f Same Sera Tested against Culture Virus f 54th Transfer and a New Culture in the 5th Transfer IV Culture virus (P.R. 8) Serum Nrmal rabbit 1-29... " swine 16-52... Swine 16-52, immune t swine influenz~... Human R. D... " D. T... " T. F... " W. M... 54th transfer (Original culture) ' k *i * Muse N. 5th transfer (New culture) Muse N. 1 I 2 2 *i ± + + *+- + + * *!+ + + + +±! + * * I 3 + prduced by a smaller number f particles f virus maintained by animal passage. This pssibility is heightened by the impressin gained that the lesins in the mice infected with culture virus, althugh f equal extent, are less intense than thse btained with passage f the virus frm animal t animal. SUMMARY The ir~ vitr cultivatin f strains f human influenza virus has been successfully cnducted thrugh a prlnged series f successive transfers. The cultivated virus has retained the antigenic and immunlgical prperties which characterized the animal passage virus frm

T. P. MAGILL AND THOMAS FRANCIS~ JR. 811 which it was derived. The culture virus is still virulent fr mice and ferrets; it is capable f inducing an active state f immunity in animals vaccinated subcutaneusly r intraperitneally; it elicits specific neutralizing antibdies in the serum f infected r vaccinated animals. The virus has been successfully cultivated t date nly in the presence f xygen; when cnditins f reduced xygenatin are impsed by the use f vaseline seal, with r withut the additin f cystein, multiplicatin f the virus is nt supprted. On the ther hand, it has been pssible t cultivate the virus in the medium f Li and Rivers in rdinary test tubes. This affrds a greatly simplified prcedure, since the interval between transfers may be prlnged. The results f neutralizatin tests with varius sera and the culture virus are presented and discussed. BIBLIOGRAPHY 1. Francis, T., Jr., and MagiU, T. P., Science, 1935, 82, 353. 2. Francis, T., Jr., Science, 1934, 8, 457. 3. Francis, T., Jr., Prc. Sc. Exp. Bil. and Med., 1935, 32, 1172. 4. Shpe, R. E., J. Exp. Med., 1931, 54, 573. 5. Burnet, F. M., Med. J. Australia, 1935, 9., 687. 6. Smith, W., Brit. Y. Exp. Path., 1935, 16, 58. 7. Li, C. P., and Rivers, T. M., J. Exp. Med., 193, 59., 465. 8. Maitland, H. B., and Maitland, M. C., Lancet, 1928, 2, 596. 9. Francis, T., Jr., and Magill, T. P., Y. Exp. ivied., 1935, 69., 55. 1. Smith, W., Andrewes, C. H., and Laidlaw, P. P., Brit. Y. Exp. Path., 1935, 16, 291. 11. Francis, T., Jr., and MagiU, T. P., Prc. Sc. Exp. Bil. and Med., 1936, 33, 64.