STUDIES ON THE VIRUS OF INFLUENZA

Transcription

1 STUDIES ON THE VIRUS OF INFLUENZA BY A. R. DOCHEZ, M.D., K. C. MILLS, AI~D YALE KNEELAND, JR., M.D. (Frm the Department f Medicine, Cllege f Physicians and Surgens, Clumbia University, and the Presbyterian Hspital, New Yrk) (Received fr publicatin, January 23, 1936) Frm the time f its discvery in 1891 until 1918 the bacillus f Pfeiffer was generally regarded as the cause f influenza; as a cnsequence f this belief nearly all f the huge vlume f investigatin dne at the time f the recent great pandemic cncerned itself with this rganism. These studies gave rise t many cnfused and cntradictry results, and ended by casting dubt upn the etilgical significance f Pfeiffer's bacillus, r any ther f the visible micrrganisms that culd be cultivated frm the respiratry tract. As a result f this uncertainty a filtrable virus was sught in cnnectin with influenza, and as early as August, 1918, Selter (1) reprted the prductin f a mild influenza-like disrder in ne f 2 vlunteers wh inhaled the filtered thrat secretins frm an active case f the disease. A few ther reprts (24) seemed t cnfirm this bservatin, but the very small number f experiments, the lack f quarantine prcedures, variatin in incubatin perid, and the fact that an epidemic was in prgress at the time, surrunded these early essays with an atmsphere f dubt. In additin, it is ntewrthy that ther bservers (5-7) failed t btain like results in a larger series f attempts, and by 1922 it is safe t say that the evidence against a filtrable virus as the cause f influenza was mre cnvincing than that in favr f it. Since 1918 there has been n great pandemic f influenza; the existence, hwever, f s called interpandemic influenza, rdinarily ccurring in winter in rather lcalized utbreaks, in which many f the cases clsely resemble the pandemic variety, has tempted investigatrs in recent years t direct their attentin again t this disease. In 1931 Lng and his cwrkers (8) reprted the appearance in chimpanzees f a febrile disease after inculatin with filtered secretins frm influenza patients; in 1932, hwever, Csta-Mandry et al. (9) failed t achieve the same result in human vlunteers with material btained during the Puert Ric epidemic f that year. Our wn studies f the cmmn cld sn led us t the belief that this prblem culd nt be separated frm that f influenza. Clinical and epidemilgical studies alike suggest that many examples f influenza during a typical utbreak s clsely resemble the cmmn cld as t be indistinguishable frm it. This has been bserved in islated cmmunities in the far nrth when infectin has been intrduced frm the utside by a single individual. A study and knwledge f bth diseases is therefre essential t the determinatin f the value f any frm 581

2 582 VIRUS OF INFLUENZA f prphylactic immunizatin. In an accmpanying paper (1) we have reprted in detail ur investigatins f the cmmn cld which cnvinced us that the primary etilgical agent f this disease is a filtrable virus. Having develped a technique fr the study f this virus, it has seemed advisable t apply this same technique t the study f interpandemic influenza. In 1933 we reprted (11) the cultivatin in vitr f a virus derived frm a patient with influenza, and the cultivatin f a secnd strain was nted in 1934 (12). In 1933 (13) Smith, Andrewes, and Laidlaw annunced that they had succeeded in initiating a virus disease f ferrets, transmissible in series, with filtered thrat secretins f influenza patients, and later n (14) bserved that the influenza virus after passage thrugh ferrets became als pathgenic fr mice. Similar bservatins have been made by Francis (15). Althugh it has nt yet been pssible t prduce influenza in human beings with this virus, nevertheless the ishtin f a number f strains frm different parts f the wrld and their serlgical identity as shwn by bth Andrewes (16) and Francis (17), and the study f the prtective pwer f cnvalescent serum by the latter, strugly suggest that these wrkers are dealing with the virus f interpandemic influenza. Recently Francis (18) has annunced that the virus btained frm muse lung can be cultivated in tissue medium under aerbic cnditins. The bject f the present paper is t reprt in detail the experiments we have perfrmed with the virus f influenza frm 1931 t Methds The methds used in islating the virus, preserving it, cultivating it, and testing fr its activity in human vlunteers, have been described in cmplete detail in a previus publicatin (1). RESULTS 1. Attempted Transfer f Influenza frm Individual t Individual.- The first experiment was perfrmed in January, 1931, and cnsisted f the intranasal inculatin f a fresh, bacteria-free filtrate f nse and thrat washings frm a patient with influenza. Case.--M. S., a student nurse free frm upper respiratry symptms fr a perid f 2 mnths, suddenly nted cugh, malaise, and mderate sre thrat; later n she develped nasal bstructin and discharge. Temperature at nset 11.6 per s, W.B.C. 7,3. She remained in bed 4 days, and returned t duty n the 6th day. During this mnth there was a rather high incidence f influenza in New Yrk City. The washing was taken 24 hurs after the nset f the disease.

3 A. R. DOCHEZ, K. C. MILLS, AND Y. KN'EELAND, JR. 583 Chart 1 Vlunteer 25. Virus: filtered washing frm M.S. Date, Observatin perid Jan Inculatin Jan Nasal bstructin. Sneezing. Nasal discharge... Cugh.. Sre thrat.. Headache. Malaise. Fever.. ± very mild, mild, mderate, marked, severe. As will be seen frm the freging chart, this subject develped very mild symptms fllwing intranasal inculatin; these were entirely referable t the upper respiratry passages, with the exceptin that cugh became marked n the day f discharge frm the hspital. Cnstitutinal symptms were absent. The type f respnse manifested by this individual indicated the presence in the filtrate f a virus similar in actin t the virus f the cmmn cld. 4 days after discharge he reappeared at the hspital with a temperature f 13 per s, cmplaining f cugh, headache, and marked prstratin. He stated that his cld became wrse immediately after discharge, and that he felt feverish the next day. He was fund t have brnchitis and sinusitis, and remained 2 weeks under treatment n the medical wards. The significance f the relapse and intensificatin f symptms is nt clear. Perhaps utside expsure caused a cmplicatin f his infectin. Vlunteer 26, wh was simultaneusly inculated with the same material, manifested n signs f infectin. On the basis f this single experiment tentative pinin nly culd be frmed that there existed in ne patient suffering frm interpandemic influenza, a virus similar in actin in an experimental individual t the virus f cmmn cld. 2. Experiments with Virus f Interpandemic Influenza Cultivated in Vitr.--Strain F was derived frm the fllwing case. In February, 1932, a telephne peratr was abruptly seized with nausea, fllwed by headache, backache, and a shaking chill. Temperature 1 day later was 13, W.B.C. 7,. She shwed slight cyansis, injected cnjunctivae,

4 i 584 VIRUS OF II~]~LUENZA sme nasal bstructin, and a dry cugh. Fever lasted 4 days, and n the 3rd day the white bld cunt was 4,8. Influenza was prevalent at the time. This patient had als a very mild chrnic cystitis which was nt altered by the superimpsed respiratry infectin. A tissue culture was initiated with material derived frm this patient in the manner already described (1). Transfers were made at 3 t 5 day intervals. In the 19th transfer f the culture, Strain F was tested n 2 vlunteers. Chart 2 Vlunteer 55 a. Virus F 19 Date, Nasal bstructin... Sneezing... Nasal discharge... Cugh... Sre thrat... Headache... Malaise... Fever... Ap 29 ~nculatin May m m ± This patient, as is seen frm the abve chart, develped a typical head cld f mre than average severity. There was an exacerbatin f symptms n the 5th day, but thrughut its curse the disease had bvius characteristics f a head cld, with mild cnstitutinal reactin and n fever. On the 3rd day the leuccytes were 9,5. Chart 3 Vlunteer 56 a. Virus F 19 Date, 193g... Apr. 29 Inculatin May Nasal bstructin. Sneezing.. Nasal discharge. Cugh... Sre thrat. Headache. Malaise... Fever. ± q- ± -k ± ± ± ± ± -4- ± Like the preceding vlunteer, this man develped symptms characteristic f a severe head cld. There was nly a mild degree f malaise, and n fever. On the 3rd day the leuccytes were 1,5.

5 A. R. DOCHEZ, K. C. MILLS, AND Y. KNEELAND, JR. 585 This experiment indicates that frm a typical example f the interpandemic frm f influenza a filtrable virus was cultivated in tissue medium. This cultivated virus when inculated intranasally int 2 human vlunteers prduced in bth vlunteers the symptms f a severe cmmn cld. The degree f cnstitutinal reactin was slight and neither f the vlunteers experienced a rise in temperature. There was n characteristic alteratin in the leuccyte cunt. In view f the similarity f the symptmatlgy t that f cmmn cld, an effrt was made t determine if infectin with the abve culture virus gave a crss immunizatin against the culture virus f cmmn cld. An attempt was first made t test the immunity f the 2 abve individuals against infectin with the hmlgus culture virus strain F. The men were auwed t g hme and readmitted t the islatin quarters after the lapse f 3 weeks when they were again inculated intranasally with strain F transfer 26. The results are shwn in the fllwing chart. Chart 4 Vlunteer 55 b. Virus F 26 Date, Nasal bstructin... Sneezing... Nasal discharge... Cugh... Sre thrat... Headache... Malaise... Fever... ] i Incu- latin 2S ] 26 ] 27 May 2 May O ' ' -4- Vlunteer 56 b. Virus F Date, May ~asal bstructin. ;neezing. ~asal discharge. ;ugh. re thrat... [eadache. ~alaise.. 'ever... ' l "1 ' ± 4- ±

6 586 VIRUS O1 ~ INFLUENZA The abve chart shws that bth vlunteers were susceptible t reinculatin with the F strain f influenza virus 3 weeks after their riginal infectin. In bth the disease prduced was a mderate head cld withut cnstitutinal reactin. Bth men declared that the symptms were quite similar t thse prduced by the first inculatin, althugh less than half as severe, an bservatin brne ut by a cmparisn f the tw charts. This experiment demnstrated that an infectin with a strain f culture virus did nt cnfer a slid immunity against reinfectin with the same strain. Since little immunity appears under the circumstances f the experiment t develp against the hmlgus strain, the methd is nt suitable fr crss immunity tests. Culture strain F was subsequently carried in vitr until Nvember, The ttal perid f cultivatin frm the time f islatin was apprximately 2 mnths. The intervals f transfer varied frm 3 t 5 days. During this perid human vlunteers were inculated frm time t time and the results f these inculatins are presented in summary in Chart 5. Chart 5 Date Vlunteer/fr. Transfer f culture Intensity and duratin f symptms Respiratry Cnstitutinal Fever 193g July Oct. Nv. ~t 1933 Jan. cl Oct. c~ Nv. lg days days -I J F., p

7 A. R. DOCHEZ, K. C. MILLS, AND Y. KNEELAND, JR. 587 The histry f culture strain F as depicted in Charts 3, 4, and 5 presents sme interesting facts. The strain f virus was islated frm a patient suffering frm a typical attack f influenza f the interpandemic frm. There was a mderately high incidence f similar clinical types f influenza at the time. The first series f vlunteers respnded t inculatin with the symptms f a severe cmmn cld with marked upper respiratry manifestatins and slight r n cnstitutinal reactin. This was fllwed by a perid during which inculatin f the culture prduced little r n symptms f any kind. During this perid a strain f the culture was transferred t Lndn where a number f experimental inculatins were made, the majrity f which gave n psitive evidence f infectin. In the series f vlunteers 64 t 7 the symptmatlgy f the experimental disease fllwing inculatin changed markedly. These individuals, after an incubatin f 24 hurs r lnger, all develped rather marked cnstitutinal symptms cnsisting f generalized aches and pains, lethargy, and anrexia. In additin t these manifestatins they suffered frm a mild degree f fever, which up t this time had never been bserved in any f the experimentally prduced upper respiratry infectins. On the ther hand the symptms f irritatin f the respiratry tract were very slight. 9 mnths after these inculatins the culture had entirely lst its Opacity t induce symptms in an inculated individual. In January, 1933, a secnd strain f influenza virus, strain T, was cultivated. A graduate nurse, free frm respiratry symptms fr the previus 3 mnths, suddenly nted general slight malaise, a sensatin f swelling in the thrat, and sme cugh. In the evening the malaise deepened, and she had chilly sensatins, pain in the back and neck, and prstratin. At the end f a restless night her temperature was 11.6 F., W.B.C. 5,8. Aside frm sme swelling f the lymphid tissue in the pharynx, there were n significant physical signs. At the end f 48 hurs the fever had subsided, and the patient made a rapid recvery. Influenza was prevalent at this time. A tissue culture was initiated with material frm this patient in the usual manner, transfers being made at 3 t 4 day intervals. The strain was first tested n 2 vlunteers in the 18th transfer. In this particular experiment an inculatin f filtered naspharyngeal washings frm a nrmal individual was made 3 days befre the actual test with strain T. The bject f this inculatin was t ascertain if a virus capable f inducing respiratry symptms might be carried by a nrmal persn during the winter mnths. This inculatin was fllwed by n signs f infectin. The results f inculatin with strain T are given in Chart 6. Chart 6 shws that bth men develped head clds, ne smewhat mre severe than the ther. Bth had an exacerbatin f symptms n the 4th day. Symptms were, fr the mst part, limited t irritatin f the upper respiratry tract, and the degree f general malaise was insignificant. Neither man had fever.

8 588 VIRUS OF INFLUENZA Chart 6 Vlunteer 73. Virus T 18 Date, Inculatin Mar. 1 I Mar Nasal bstructin... Sneezing. Nasal discharge... Cugh.. Sre thrat.. Headache. Malaise... Fever i Vlunteer 74. Virus T 18 Date, Nasal bstructin... Sneezing... Nasal discharge... Cugh... Sre thrat... Headache... Malaise... Fever... Mar :{ I I,~ "ll ll ' ± 4-4- ± Strain T was next tested in the 8th transfer; the results are given in Chart 7. Chart 7 Vlunteer 8. Virus T 8 Inculatin Date, Oct. 27 ~ Oct Nv. l 3 Nasal bstructin. Sneezing. Nasal discharge... Cugh.. Sre thrat., Headache. Malaise. Fever. -4-

9 A. R. DOCHEZ, K. C. MILLS, AND Y. KNEELAND, JR. 589 Chart 7--Cncluded Vlunteer 82. Virus T 8,ate, Incu- I latin Oct. 27 ] Oct Nv [asal bstructin. neezing. [asal discharge... ',ugh.. re thrat... [eadache. {alaise... 'ever _ Vlunteer 81. Virus T 8 Date, 193J... Oct Nv Nasal bstructin... Sneezing. Nasal discharge... Cugh... ;re thrat... Headache. Malaise... Fever. These results shw that strain T was still capable f prducing infectin in the 8th transfer, the symptms which it prduced being chiefly thse f upper respiratry irritatin. Vlunteer 81 shwed a Chart 8 Vlunteer 88. Date, Feb. 23 Nasal bstructin... Sneezing... Nasal discharge... Cugh... Sre thrat... Headache... Malaise... Fever... Virus T113 and 114mlxed Incu- ] latin I Feb. d ~'I ax. 1 i 3 i

10 59 VIRUS OF INFLUENZA mderate degree f malaise. N elevatin f temperature ccurred in this grup. Strain T was tested fr the last time n ne vlunteer wh was inculated with a mixture f the ll3th and 114th transfers f the culture. The result is shwn in Chart 8. Strain T, therefre, shwed sme infective pwer after mre than 1 transfers f the culture. N subsequent tests were made. T summarize ur experience with this strain f virus derived riginally frm a mild case f influenza, strain T prduced in the 6 vlunteers inculated symptms fr the mst part referable t infectin f the upper respiratry tract and f the type bserved in cmmn cld. The degree f cnstitutinal reactin was slight and there was n fever. In March, 1934, a furth strain f influenza virus, strain R, was cultivated frm a patient suffering frm influenza which ccurred at that time as a lcal epidemic in the Pelham Hme. This establishment huses a ppulatin f 35 girls, all f whm are rheumatic fever subjects. In March, 1934, a serius epidemic f influenza tk place in the Hme, apparently intrduced by the ck. Cburn and Pauli (19) have reprted the clinical aspects f this utbreak in detail, tgether with a bacterilgical and immunlgical study f the individual cases. The main clinical features f the disease at the Pelham Hme were: rather sudden nset, fever f abut 14 fr 3 days, with marked prstratin, nausea, vmiting, and insignificant symptms f upper respiratry irritatin. As the epidemic prgressed a variatin in the type f respnse tk place, s that the intermediate cases shwed increasing symptms f respiratry irritatin and decreasing fever. The last cases f the disease were afebrile and resembled the cmmn cld. Leucpenia was nt bserved during the utbreak. Strain R was derived frm a typical example f the disease at the beginning f the epidemic. It was cultivated in tissue medium fr 18 transfers and then tested in 3 vlunteers. The results are given in Chart 9. These prtcls shw that the 18th transfer f strain R f influenza virus prduced quite different effects in 3 vlunteers inculated with the same dsage under identical cnditins. Vlunteer 93 experienced the symptms f a cmmn cld f mderate intensity which lasted 3 days. N. 94 had symptms f a severe cld lasting 5 days, and in additin, n the first 4 days there was a marked cnstitutinal reactin cnsisting f prstratin, dizziness, lss f appetite, and pain in the neck. On the night f May 29 he vmited several times. There was n fever.

11 Chart 9 Vlunteer 93. Virus R 18 Date, MAY25 ~nculatin 3 31 June Nasal bstructin. Sneezing. Nasal discharge... Cugh.. Sre thrat.... Headache... Malaise. Anrexia... Vmiting... Prstratin.... Dizziness... Fever.. 4- Vlunteer 94. Virus R 18 Date, June i 3 4 Nasal bstructin. Sneezing... Nasal discharge... Cugh.. Sre thrat... Headache. Malaise. Anrexia... Vmiting. Prstratin... Dizziness.... Fever Vlunteer 95. Virus R 18 Date, June Nasal bstructin. Sneezing.... Nasal discharge... Cugh.... Sre thrat.... Headache.... Malaise... Anrexia.... Vmiting.... Prstratin... Dizziness... Fever p.. : ± ± 1 Nausea L il ± Weakness 591

12 592 VIRUS OF 1-N]~LUENZA The effect f inculatin in vlunteer 95 was striking; while cryzal symptms were nt prminent after the first day, severe cnstitutinal manifestatins develped which did nt disappear entirely fr 6 days, and a cugh which was still present n discharge frm the hspital a week after inculatin. On the 2nd day f his disease he presented an appearance like that f influenza; he was prstrated and cmplained f aching pains, chilly sensatins and sweating, marked dizziness, nausea, and vmiting. His eyes were suffused, face flushed, thrat diffusely red. Lungs were clear. The muth temperature was 11 F. Leuccytes were 1,9. Except fr the fact that his fever lasted nly a day, the clinical picture was like that seen in interpandemic influenza f average severity. The vlunteer declared that the experimental disease was very similar t, thugh smewhat less severe than, an attack f influenza he had experienced in 1918 while in the army. Strain R was tested fr activity in 5 vlunteers in later transfers f the culture; the results f these tests are briefly summarized in Chart 1. Chart 1 Date Vlunteer N. Transfer f culture Intensity and duratin f symptms Respiratry Cnstitutinal Fever 1934 Sept. Oct R 53 R 53 R63 R63 R 63 3 days 4-2 " 3 " Chart 1 shws that f 5 inculatins int human vlunteers f later transfers f the culture strain R a mild cryza resulted in 3 instances. In summary, strain R was islated frm a patient during an utbreak f influenza which ccurred in a small semi-islated ppulatin. When tested in the 18th transfer this culture virus gave rise t ne infectin resembling clinical influenza, t ne "grippy" cld, and t ne cryzal type f cld. Subsequent inculatins f the 53rd and 63rd transfers resulted in mild clds f the cryzal type in which there were n cnstitutinal symptms. In Octber and Nvember, 1935, tw additinal strains, Ra and T,

13 A. R. DOCIIEZ, K. C. MILLS, AND Y. KNEELAI~D, Jl~. 593 were cultivated frm tw examples f what appeared t be the spradic type f influenza. Strain Ra was btained frm a 41 year ld man with a past histry f maxillary sinusitis. 4 days befre admissin he caught cld and felt feverish; he had nasal discharge, dry cugh, and increasing malaise. The day befre admissin his temperature was said t have been 14. On admissin it was 11, and became nrmal 2 days later. W.B.C. 5,9. X-ray examinatin was suggestive f sinusitis, and 6 days after admissin the right antrum was irrigated and an imprvement f the patient's cnditin resulted. A naspharyngeal washing was btained n the day f admissin, and a culture initiated in the usual manner. The final diagnsis f this patient's cnditin was acute sinusitis. Strain T was btained frm a patient with the fllwing histry: A 45 year ld American husewife, after expsure at the huse f a friend t a case f what was called influenzal pneumnia, suddenly nticed chilly sensatins, and extreme prstratin. Her temperature was fund t be 13 F. The next day she was admitted t the hspital. There were n symptms f respiratry irritatin. Her nly cmplaints were feverishness, great prstratin, and generalized aching pains. Physical examinatin shwed an acutely ill wman with sme helitrpe cyansis and a slightly red thrat. W.B.C. 3~25. Fever lasted 3 days, the highest temperature being 14. W.B.C. remained belw 7, fr 2 weeks and the patient remained in the hspital 27 days n accunt f extreme asthenia which disappeared very slwly. During mst f this perid the bld pressure was lw, and fr the first week f her hspital stay capillary fragility was increased as was indicated by a psitive turniquet test. Althugh n epidemic was in prgress at the time, the symptms frm which this individual suffered exactly crrespnded t thse f uncmplicated influenza f the pandemic variety. A naspharyngeal washing was btained 48 hurs after the nset f the disease and cultivated in tissue medium fr 13 transfers befre inculatin f human vlunteers. The vlunteers used in the test f culture T had a few days previusly been inculated with culture Strain Ra, which had prven inactive. The results f inculatin with bth Strain Ra and T are presented in Chart 11. These prtcls shw that inculatin f strain Ra prduced n infectin in either f the 2 vlunteers. Hwever, bth were fund t be susceptible a few days later t infectin with strain T. In view f the fact that the final diagnsis f patient Ra's cnditin was acute sinusitis, it is pssible that n filtrable virus was present in the respiratry passages. The type f infectin prduced by inculatin f strain T was different in the 2 vlunteers tested. In vlunteer 12, cnstitutinal symptms--pains abut

14 594 VIRUS OF INI~LUENZA Chart 11 Vlunteer 12 Virus Ra 8 and 9 Virus T 13 Date, Nasal bstructin.. Sneezing... Nasal discharge... Cugh... Sre thrat. Headache.. Malaise... Fever... Inculatin 24 N2~v" l Incu [ latin 27 2s I N2 gv- 3 Dec, _, 2 m Vlunteer 121 Virus Ra 8 and 9 Virus T 13 Date, Nv Dec Nasal bstructin.. Sneezing... Nasal discharge... Cugh... Sre thrat.. Hcadachc.. Malaise... Fever. I! i 4- : the head and ears, dizziness, insmnia, and anrexia--were prminent features f the experimental disease. In additin, symptms f upper respiratry irritatin and severe cugh were present. There was n fever. In vlunteer 121 the symptms resulting frm inculatin were f the cryzal type accmpanied by mderately severe cugh and sre thrat, and there was little r n cnstitutinal reactin. Thrughut the curse f these studies careful bservatin f the bacterial flra f the naspharynx f inculated vlunteers was maintained. In accrdance with ur usual prcedure individuals harbr-

15 A. R. DOCHEZ, I(. C. MILLS, AND Y. I(N'EELAND, JR. 595 ing Pneumcccus Types I, II, r III, r S. hemlyticus were nt used fr experimental infectin. Hwever, certain individuals thrughut the perid f an experiment harbred in their thrats pneumccci f types ther than thse mentined and frequently cnsiderable numbers f H. influenzae. The presence f such rganisms during the curse f an experimental infectin did nt seem, as far as culd be determined by bservatin, t alter the severity r character f the clinical manifestatins when cmpared with the experimental disease in individuals free frm pneumccci r H. influen~ae. We have previusly bserved that during a spntaneus r experimental cld in chimpanzees (2), when H. influen~ae is present in the naspharynx f these animals a shift frm the R frm f this rganism t the S frm may take place. N such change f type f H. influen~ae, thugh carefully lked fr, was bserved in human beings. In a number f instances the virulence f the pneumcccus present was tested by muse inculatin bth befre and after inculatin. N change in virulence was bserved. It is true, hwever, that cmplicatins such as purulent sinusitis and severe brnchitis were nt bserved and it is pssible that the cnditins under which the experiments were perfrmed diminished the liability t such cmplicatins. DISCUSSION In this paper are presented the results f ne attempt t transfer influenza frm ne human being t anther by direct inculatin f a bacteria-free filtrate f naspharyngeal washings and f five attempts t cultivate in tissue medium a filtrable virus frm human cases f influenza bth f the epidemic and spradic type. As a cnsequence f this study the cnclusin can be drawn that there is present in filtered naspharyngeal washings derived frm an acute case f influenza a nn-bacterial filtrable agent which is capable f prducing in human vlunteers infectin f the upper respiratry tract and that this agent can be successfully prpagated in a medium cntaining minced chicken embry. The cultivated agent prduces n visible change in the medium in which it grws nr can visible bacterial frms be demnstrated by. subculture r by micrscpic study. It belngs t the class f filtrable viruses. In all, 39 human vlunteers have been experimentally inculated

16 596 VIRUS OF INFLLrENZA with this filtrable agent. The utstanding utcme f these inculatins has been that in every instance but ne the inculatin, when psitive, whether f fresh filtered washings r f the cultivated virus, has resulted in the prductin f an infectin mre nearly resembling the cmmn cld than human influenza. A significant explanatin f these results cannt be given at the present time. It is, f curse, well knwn that a large number f upper respiratry infectins ccurring during a typical epidemic f influenza resemble in character the cmmn cld. Furthermre, in utbreaks f respiratry infectin in clnies in the far nrth which riginate frm a single surce, types f infectin resembling bth influenza and cmmn cld are present at the same time. Other explanatins f the predminant ccurrence f infectins resembling cmmn cld in the experimentally inculated individuals may be ffered. It is nt unlikely that the cnditins f cultivatin f the virus result in a diminutin f virulence s that the infectin fllwing inculatin manifests symptms f less severity. Mrever, the imprtance f virulent secndary rganisms in the prductin f the typical picture f human influenza is nt understd at the present time, and the absencef such rganisms frm the naspharynx f the inculated vlunteers may have influenced the character f the symptmatlgy. In additin, the fact that all vlunteers during the perid f infectin were maintained in the favrable and prtected envirnment f a private hspital suite may als have influenced the character f the experimental disease. Finally, there is n way at the present time f differentiating cnclusively the virus f cmmn cld frm that f influenza. That these tw viruses are clsely related in their manifest activity is extremely likely and that they are clse t ne anther in bilgical nature seems prbable. On the whle, experimental infectins induced in human beings by inculatin f the filtrable virus cultivated frm influenza have been mre severe in their symptmatlgy than a cmparable series f infectins arising frm inculatin f the cultivated virus f the cmmn cld. Furthermre, the tendency t exhibit a mre prnunced cnstitutinal reactin after inculatin f influenza virus is mre cnspicuus. These phenmena appear in cnnectin with three f the viruses studied--designated strains F, R, and T. A study f the

17 A. 11. DOCHEZ, K. C. MILLS, AND. KNEELAND, JR. 597 case histries f the patients frm which these three were derived indicates that they apprached mre clsely the epidemic type f influenza than did the tw (strain T and the filtrate experiment) in which the virus btained was indistinguishable frm the cmmn cld virus. In nly ne (vlunteer 121) f 39 vlunteers inculated has the clinical picture f human influenza been reprduced. This was effected by the virus (R) frm the Pelham Hme utbreak--an utbreak which aside frm its purely lcal character cnfrmed t the epidemic pattern. It is interesting that f 2 additinal vlunteers inculated at the same time and with the same material and maintained under identical cnditins, ne develped a mild cryzal type f cmmn cld and the ther a cld f the s called grippy variety. Lastly, ne attempt was made t cultivate a virus frm a case f upper respiratry disease with high fever which appeared t be acute sinusitis rather than influenza. N virus was btained frm this case in an attempt made 4 days after the nset f symptms. SUMMARY AND CONCLUSIONS i. Evidence is presented indicating the presence f a filtrable virus in the naspharyngeal secretins f individuals suffering frm influenza. 2. An attempt t transfer influenza frm ne human being t anther by means f filtered naspharyngeal washings resulted in the prductin in the inculated vlunteer f a cmmn cld. 3. A filtrable agent has been cultivated in tissue medium frm the filtered naspharyngeal washings f patients with influenza. 4. Inculatin f the cultivated virus int human vlunteers results fr the mst part in the prductin f a severe cmmn cld with a tendency t prnunced cnstitutinal reactin. 5. In ne instance fllwing inculatin f culture virus an infectin clinically resembling influenza has been prduced. 6. The mre clsely the surce f the virus apprached the type f epidemic influenza, the mre likely the virus was t prvke cnstitutinal symptms. 7. The presence f certain pathgenic bacteria in the upper respiratry tract f inculated individuals was nt bserved t mdify the curse r character f the experimental infectin.

18 598 VIRUS OF INFLUENZA 8. On prlnged cultivatin the virus lses the capacity t infect human vlunteers. BIBLIOGRAPHY 1. Selter, H., Deutsch. reed. Wch., 1918, 44, Niclle, C., and Lebailly, C., Ann. Inst. Pasteur, 1919, 33, Leschke, E., Berl. Klin. Wch., 1919, 56, Yamanuchi, T., Sakakami, K., and Iwashima, S., Lancet, 1919, 1, Kruse, W., Manch. reed. Wch., 1918, 65, Friedberger, E., and Knitzer, P., Med. Klln., 1919, 15, Rsenau, M. ]., J. Am. Med. Assn., 1919, 73, Lng, P. H., Bliss, E. A., and Carpenter, H. M., J. Am. Med. Assn., 1931, 97, Csta-Mandry,., Mralcs-Oter, P., and Suarcz, J., Puert Ric J. Trp. Med. and Pub. Health, 1932, 8, Dchez, A. R., Mills, K. C., and Kneeland, Y., Jr., J. Exp. Med., 1936, 63, Dchez, A. R., Mills, K. C., and Kneeland, Y., Jr., Prc. Sc. Exp. Bil. and Med., 1933, 3, Dchez, A. R., Mills, K. C., and Kneeland, Y., Jr., Prc. Sc. Exp. Bil. and Med., 1934, 32, Smith, W., Andrewes, C. H., and Laidlaw, P. P., Lancet, 1933, 9., Andrewes, C. H., Laidlaw, P. P., and Smith, W., Lancet, 1934, 2, Francis, T., Jr., Science, 1934, 8, Andrewes, C. H., Laidlaw, P. P., and Smith, W., Brit. J. Exp. Path., 1935, 16, Francis, T., Jr., Prc. Sc. Exp. Bil. and Med., 1935, 39., Francis, T., Jr., and Magill, T. P., Science, 1935, 89., Cbum, A. F., and Pauli, R. H., J. Exp. Med., 1935, 69-, Dchez, A. R., Mills, K. C., and Kneeland, Y., Jr., Prc. Sc. Exp. Bil. and Med., 1932, 3, 314.