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Studies of Adenoviruses APC * in Volunteers JOSEPH A. BELL, M.D., Dr.P.H., F.A.P.H.A.; THOMAS G. WARD, M.D., Dr.P.H.; ROBERT J. HUEBNER, M.D., F.A.P.H.A.; WALLACE P. ROWE, M.D.; R. SUSKIND, M.D.; and RALPH S. PAFFENBARGER, Jr., M.D., Dr.P.H. GERALD S-CC and CC inoculums and only An outstanding feature of this re- homologous inmmunity to illness from search that wiiU be of immediate the ARD interest to aU in public health is inoculum. It was concluded the report on a field trial of APC that at least two agents, presumably vaccine which furnishes the back- viral, causing ground data and the basic essen- were acute respiratory tials for the use of such a vaccine.diseases of unknown etiology. In 1953, Rowe, et al.,3 reported the discovery of adenoid degenerating In 1947, the U. S. Army Commission agents later classified 4' 5 as the ade- on Acute Respiratory Diseases 2 re- noidal-pharyngeal-conjunctival (APC) ported experimental transmission to group of viruses and recently designated human beings of etiologically undif- adenoviruses. This group of viruses is ferentiated acute respiratory illnesses. characterized by production of unique Volunteers were inoculated by spraying cytopathogenic changes in cultures of the nasopharynx with 10 ml of bacteria- epithelial cells and by nonpathogenicity free filtrates of nasopharyngeal excre- for ordinary laboratory animals. They tions collected from (a) a donor ill with are heat labile, filterable, and resistant an acute febrile respiratory disease to antibiotics and ether, and have (ARD), and (b) from donors ill with soluble, group-specific, complement-fix- severe ("S-CC") and mild ("CC"), ing antigens which are not type-specific. nasopharyngeal catarrh. Inoculation Twelve human and two simian immuno- was followed by minor respiratory logically distinct serotypes have been illnesses chiefly manifest by nasopharyn- delineated to date by type-specific geal catarrh at from five to six days in neutralization tests.6 Types 1 through the ARD group and from one to two 6 have been found in human tonsil and days in the S-CC and CC groups. adenoid tissue removed surgically from Reinoculation of both groups with persons without acute febrile illness. homologous and heterologous materials Types 1 through 7 have been found on showed no immunity to illness from the the pharynx or conjunctiva of human beings with acute, febrile, respiratory * On May 25, 1956, the investigators respon- illness. A single strain of Type 8 has sible for using the names Adenoid Degenera- tive (AD) viruses, Adenoidal Pharyngeal been recovered from the eye of a human Conjunctival (APC) viruses, Respiratory Ill- being with epidemic keratoconjunctivi. ness (RI) viruses, and Acute Respiratory tis and serologic evidence in 25 cases Disease (ARD) viruses to designate a new suggests an etiologic relationship.7 Only group of respiratory viruses agreed to use single strains of other known types have henceforth the term "adenovirus" in lieu of former names.' been recovered to date. The Maryland Studies were supported in In 1954, Hilleman, et al.,8 reported part by grants from the Research Grants Division, U. S. Public Health Service and that a newly isolated virus (RI 67) was from the Common Cold Foundation. associated with ARD in military re- 1130 ADENOVIRUSES VOL. 46 1131 cruits. RI 67 virus was found to be Types 1 through 6 were used. The Type 4 of the virus group previously inoculums consisted of (a) human nasal reported by Rowe and Huebner and re- washings known to contain Type 3 or 4 cently designated adenovirus. In 1955, virus; (b) Types 1, 2, 5, and 6 virus Ginsberg, et al.,9 reported that serums grown in cultures of human embryos or from the donor and recipients of ARD tonsil or adenoid cells; (c) Types 1, 2, inoculums used in Army Commission 3, and 4 virus grown in HeLa cells; (d) studies reported in 1947 showed a rise Types 1, 4, and 5 virus grown in in neutralizing antibody titer against monkey kidney cells; and (e) control RI 67 virus (APC Type 4) (Adenovirus material consisting of virus-free tissue- Type 4). culture maintenance fluid. Inoculums In August, 1953, the authors began a were administered by intranasal instilla- series of studies in volunteers to deter- tion by swabbing the oropharynx, by mine if, and under what conditions, inhalation of atomized suspensions, by various types of adenovirus could in- swabbing the palpebral conjunctiva, or duce human illness. To date 13 differ- by dropping suspensions into the con- ent studies involving 654 volunteers junctival fornix. Volunteers were also have been carried out. It was found inoculated intramuscularly and intra- that the intranasal instillation of virus dermally with heat- or formaldehyde- was followed occasionally by minor inactivated Type 3 adenovirus grown in respiratory illness which could not be monkey kidney cells. attributed to the virus inoculated. On All inoculums contained penicillin the other hand, the swabbing of virus and streptomycin, and were bacteriolog- on the conjunctiva induced readily ically sterile when tested on blood agar recognizable illness.10 Such induced and thioglycollate broth. Similarly, all illness was associated with infection as monkey kidney culture inoculums and indicated by a rise in complement-fixing the seed cultures used for preparing antibodies or by virus isolation on the HeLa culture inoculums, were tested as fifth to ninth postinoculation day. Both described previously in laboratory ani- induced infection and illness were asso- mals 10 for possible contaminating, non- ciated with susceptibility as indicated bacterial pathogens. In order to insure by the absence of preinoculation homol- complete freedom from viable tissue ogous neutralizing antibodies. It was cells, fluids were removed after all cells also found that intramuscular inocula- had demonstrated cytopathogenic ef- tion of an inactivated virus vaccine fects; these fluids were frozen at would induce neutralizing antibodies, - 200 C for at least two weeks, then and persons with such induced anti- thawed, centrifuged at 2,000 or 4,000 bodies were protected against challenge- rpm for 20 minutes, filtered through a induced infection and illness to the same extent as persons with naturally ac- Drs. Bell, Huebner, Rowe, Suskind, and quired neutralizing antibodies.11 This Paffenbarger are with the U. S. Department report reviews these studies and details of Health, Education, and Welfare, Public Health Service, National Institute of Allergy those not previously published. and Infectious Diseases, Laboratory of Infec- tious Diseases, Bethesda, Md. Dr. Ward is with the Lobund Laboratories, Notre Dame Methods of Study University, Notre Dame, Ind. (formerly at Johns Hopkins University). Studies were carried out in the Fed- This paper is an extension of the discussion presented before a Joint Session of the Epi- eral Industrial Reformatory, Chillicothe, demiology and Laboratory Sections of the Ohio, and the Maryland State Reforma- American Public Health Association at the tory, Breathedsville, Md. Adenovirus Eighty-Third Annual Meeting in Kansas City, Mo., November 16, 1955. 1132 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH UF sintered glass or Selas 0.03 filter, struction. In the early Maryland studies and refrozen until used. an arbitrary method of scoring clinical Usually, the study of a new virus findings served as an index of acute type or a new route of inoculation was illness.12 In the later studies, tabula- initiated in small groups at the Mary- tions included any manifestation of ill- land Reformatory and these were fol- ness that persisted for two or more lowed by confirmatory studies of larger consecutive days and was noted inde- groups at the Federal Reformatory. pendently by at least two observers. Study subjects were white and Negro male volunteers generally 21-29 years Pescription of Individual of age. During the course of observa- Studies and Results tion, neither the observers nor partici- pants knew which person received test Early Maryland Studies M 8/53, M 1/18/54, or control inoculums, or which inocu- M 1/25/54, M 5/54, and M 6/54-Infranasal lated site received infective material. lnsfillation of Adenovirus Types 1, 2, 4, 5, and 6 Test and control subjects were selected Each study at the Maryland Reforma- by random sampling to insure compara- tory (Study Series M) comprised 20 bility among groups, except when sus- volunteers who were often not isolated ceptibles were selected on the basis of prior to inoculation but always kept un- preinoculation antibody titers. Blood der hospital group isolation during the serums were collected just before inocu- observation period. The five early lation and again some three weeks later. Maryland studies were exploratory in The laboratory methods used for virus nature and included only 20 controls, culture and identification and for sero- since the chief objective was to observe logic complement-fixation and neutrali- if recognizable illness or infection would zation tests have been described in follow intranasal inoculation of a 1 ml previous papers.3.5 Prior to inocula- dose of suspensions containing adeno- tion, participants were examined medi- viruses. Table 1 summarizes the results cally to eliminate those with poor health of these studies; it shows the number of or acute illness. volunteers receiving various types of After test and control materials were inoculums and the number developing inoculated, participants were examined illness and complement-fixing anti- and temperatures taken almost daily bodies, together with their preinocula- throughout the period (usually ten tion homologous neutralizing antibody days) when illness was anticipated. status. Usually these examinations were made Minor respiratory illness, classified independently by two or more of the by an arbitrary scoring method but authors. The procedure was to record manifest chiefly by nasopharyngeal objective findings first and then elicit catarrh, occurred from two to five days subjective complaints and symptoms. after virus inoculation in 35 per cent The eyes were examined for conjunc- and 36 per cent respectively, of volun- tivitis; the nasal passages for inflamed teers without or with preinoculation or edematous mucosa, serous or puru- neutralizing antibodies, and in a nearly lent discharge, and airway obstruction; equal 40 per cent of the 20 controls and the mouth and throat for inflamma- who received virus-free material. On tion, exudate, and lymph follicle hyper- the other hand, a postinoculation com- trophy. Complaints were then elicited, plement-fixing antibody rise to various followed by inquiry as to headache, homologous and heterologous antigens cough, malaise, chilliness, feverishness, occurred in 65 per cent, 47 per cent, and sore throat, rhinorrhea, and nasal ob- 0 per cent of these respective groups. It ADENOVIRUSES VOL. 46 1133 Table 1-Summary of Early Maryland Studies-Number of Volunteers with Illness by Preinoculation Neutralizing Antibody Titer and Complement-Fixing Antibody Response According to Type and Source of Adenovirus Inoculated Intranasally Preinoculation Homologous N. Ab. Titer Inoculum Total No. N.Tse o No. Tested For < 1:4 1:4 Adenovirus N. Ab. and Type Source * Vol. Ill. C. F. Rise Vol. Il. C. F. Rise Vol. Ill. C. F. Rise 1 Ad 10 7 9 4 3 2 5 3 2 2 Em 9 4 7 2 2 2 5 1 3 1 and 2 Ad 11 4 7 2 1 2 5 1 4 4 NWN 10 0 10 4 0 2 6 0 2 4 NWI 10 0 9 4 0 1 5 0 1 5 Ad 13 5 13 5 1 5 8 4 1 2 and 5 Ad 10 8 7 1 1 1 6 5 5 6 Tn 7 2 6 1 0 0 5 2 3 Subtotal 80 30 68 23 8 15 45 16 21 Per cent (38) (35) (65) (36) (47) None Maint. Fluid 20 8 None (0/20) had a C. F. Rise (Per cent) (40) * Ad, Em, and Tn = virus inoculated direct from tissue culture of human adenoid, embryo and tonsil cells. NWN = pooled human nasal washings known to contain naturally occurring Type 4 virus plus normal rabbit serum. NWI = same as NWN except immune rather than normal rabbit serum added. may be noteworthy that illness followed enced the results. Hence, investigations inoculation of all viruses except Type 4. were begun at the Federal Reformatory The Type 4 inoculum differed from in Chillicothe, Ohio (Study Series C) others in that it consisted of pooled hu- using larger groups of volunteers, in- man nasal washings known to contain cluding a noninoculated control group, naturally occurring virus plus rabbit and using better standardized doses of serum; other inoculums were culture- virus grown in HeLa and monkey kid- grown viruses without rabbit serum. ney cell cultures. Though not shown in the table, illness occurred in 15 (42 per cent) of 36 Study C-3/54--Intranasal Instillation of volunteers with induced infection (four- Adenovirus Types 1, 2, and 3 fold rise in CF antibodies) and in nine The first Chillicothe study (in Study (28 per cent) of 32 without evidence Series C) comprised 267 volunteers of infection. This difference is not made available by the reformatory staff. statistically significant. Thus the minor Participants were not isolated but con- respiratory illnesses observed could not tinued their routine activities except for be attributed to the viruses inoculated, daily visits to the hospital during the despite serologic evidence of infection. study. Forty volunteers, 18-20 years The number of volunteers observed in of age, served as a noninoculated con- these early studies with group hospital trol group and were observed in the isolation was comparatively small, and same manner as other participants. The the relative amounts of virus inoculated remaining 227 volunteers, 21-29 years were not well standardized. Also, it of age, were distributed at random into was recognized that psychological fac- five groups comprising 41, 44, 50, 50, tors, allergies, or primary irritations and 44 individuals. The five groups from the inoculums could have influ- were found to be nearly equal in distri- 1134 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH bution of attributes such as age, race, tibodies. Rises were more frequent in work site, and histories of prior respira- volunteers receiving tissue-culture-grown tory illnesses, thus confirming their ran- virus than in those receiving nasal wash- dom selection. Aside from age, the ings; presumably the latter inoculum noninoculated group had a similar dis- contained smaller amounts of virus. No tribution of these attributes. biologic significance can be attributed Inoculated volunteers had 1 ml of to the difference in frequency of comple- inoculum instilled in their nostrils. The ment-fixing antibody rises in volunteers first, second, and third groups received receiving Types 1, 2, and 3 virus since HeLa-cell-grown adenovirus, Types 1, only Type 1 virus antigen was used in 2, and 3, respectively. The fourth group, these tests. Previous studies 4 showed an inoculated control group, received that Type 1 antigen gave an > fourfold virus-free tissue-culture maintenance complement-fixing antibody response in fluid. The fifth group received a pool only 76 per cent of persons infected with of three human nasal washings known other virus types. to contain Type 3 adenovirus. The Tables 3 and 4 show the percentage sixth group was the noninoculated con- occurrence of various illness manifesta- trol group mentioned above. tions by various attributes. The chief Table 2 indicates that infection fol- objective finding and subjective symp- lowed inoculation as there was a rise in tom was nasal obstruction or discharge. the postinoculation complement-fixing Illness began on the second to fifth day antibody titer. This infection correlated following inoculation and subsided on with susceptibility as it was more fre- the seventh to tenth day. A few of the quent in persons without than with de- volunteers had temperatures above tectable preinoculation neutralizing an- 100° F and were hospitalized for close Table 2-Study C-3/54-Number of Volunteers with Complement-Fixing Antibody Response by Preinoculation Neutralizing Antibody Titer According to Type and Source of Adenoviru Inoculated Intranasally (Number with C. F. Response/Number of Volunteers) Preinoculation Homologous N. Ab. Titer < 1:4 > 1:4 No Test Total Per Per Per Per Adenovirus Study cent cent cent cent Type * Group C. F. C. F. C. F. C. F. Inoculated No. Rise No. Rise No. Rise No. Rise 1 (HeLa) I 9/9 16/27 0/0 2 (HeLa) II 8/9 25/36 69 3 (HeLa) 12/28 3/5 23/42 55 III 8/13 13/34 1/3 22/50 44 Subtotal 25/31 81 41/89 46 4/8 50 70/128 55 3 (Nasal Wash.) V 2/14 14 2/26 8 0/1 .. 4/41 10 None (Maint. Fl.) IV 1/81 1 1/81 1 (Not Inoc.) VI Total 27/45 60 43/115 37 5/90 6 75/250 t 30 * (HeLa) indicates virus grown in HeLa tissue-culture cells. (Nasal washing) indicates a pool of human nasal washings known to contain naturally occurring Type 3 virus. t Seventeen volunteers not tested for C. F. antibody rise. ADENOVIRUSES VOL. 46 1135 Table 3-Study C43/54-Pereentage of Volunteers Tested Having Various Indexes of Illness According to Preinoculation Neutralizing Antibody Titer and According to Postinoculation Complement-Fixing Antibody Response Virus Inoculated Groups I, II, III, and V Preinoculation Homologous Rise C. F. Anti- N. Ab. Titer bodies > 4 Fold Nonvirus Inocu- Illness lated Control Indexes < 1:4 > 1:4 Yes No Groups IV and VI Volunteer's Opinion 41 35 32 38 27 Subjective Symptoms 43 39 32 43 24 Cough 4 2 3 3 4 Nasal Discharge or Obstruction 41 35 30 40 22 Sore Throat 2 5 5 3 1 Objective Findings 39 34 41 31 32 Nasal Discharge or Obstruction 33 28 34 25 31 Nasal Inflammation 13 9 11 9 3 Throat Signs 7 3 7 3 4 Number of Volunteers 46 122 74 95 90 Total Tested 168 169 observation. All participants had chest mediately preceding, during, or follow- roentgenograms before and after virus ing the period of observation. challenge; these showed no evidence of The Chillicothe study, utilizing larger acute pathology attributable to the groups of volunteers, including inocu- inoculum. Tables 3 and 4 show that lated and noninoculated controls, larger, illness occurred in each study group, better standardized doses of virus, and but the frequencies of manifestations more meticulous clinical observation, were not significantly related to prein- led to the same conclusions as the early oculation neutralizing antibody status, Maryland studies. Minor respiratory to rises in complement-fixing antibody illness followed inoculation, but could titer, to type of virus inoculated, or even not be attributed to the viruses inocu- to whether virus was inoculated. This lated, despite serologic evidence of in- surprising finding prompts reiteration fection. that the illness manifestations tabulated represented only those persisting for two Study C-7/54-Insfillation of Atomized or more days as recorded by two or Suspension of Adenovirus Types 3 and 5 more investigators examining independ- Failure to produce definite illness at- ently. Furthermore, each investigator tributable to inoculation of virus by the recorded objective findings before elicit- intranasal route in all previous studies ing subjective symptoms, and without led to a study in which virus was in- reference to his findings at previous haled. Each volunteer inhaled 1 ml of examinations. The volunteer was never an atomized suspension of HeLa-cell- informed of the examiners' findings. grown virus or control material. A total There was no respiratory disease out- of 50 volunteers were distributed into break among the 1,500 or more other four groups according to their neutraliz- persons at the Chillicothe institution im- ing antibody status. Group I, compris- 1136 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH Table 4-Study C-3/54-Percentage of Volunteers in Each Study Group Having Various Indexes of Illness Total Study Group I II III IV V VI Number No Virus Type 3 of Illness Adenovirus Type 1 Type 2 Type 3 Maint. Nasal Per Volun- Indexes Type Inoc.* HeLa HeLa HeLa Fluid Wash. Uninoc. cent teers Volunteer's Opinion 39 21 38 28 43 25 33 87 Subjective Symptoms 39 21 38 26 52 22 33 89 Cough 5 2 2 6 2 2 3 9 Nasal Discharge or Obstruction 37 21 32 22 50 22 31 82 Sore Throat 2 2 6 2 5 0 3 8 Objective Findings 37 33 28 34 43 30 34 91 Nasal Discharge or Obstruction 32 33 20 34 34 28 30 80 Nasal Inflammation 7 5 12 4 14 2 7 20 Throat Signs 7 5 4 6 2 2 4 12 Number of Volunteers 41 42 50 50 44 40 267 * HeLa indicates virus grown in HeLa tissue-culture cells. Nasal washins indicates human nama wa&hinns known to contain virus. ing 17 volunteers without neutralizing ficiently ill for hospital observation. Pre- antibodies to Type 3 adenovirus, re- and postinoculation chest roentgeno- ceived Type 3 virus. Group II, com- grams showed no changes. Similar to prising 14 volunteers with neutralizing previous studies where virus was inocu- antibodies to Type 3 virus, similarly lated into the nose, illness followed received an aliquot of the Type 3 inoculation but could not be attributed inoculum. Group III, comprising seven to the virus inoculated as it was not individuals without neutralizing anti- significantly related to the presence or bodies to Type 5 virus, similarly re- absence of preinoculation neutralizing ceived Type 5 virus. Group IV, antibodies. In contrast to previous comprising 12 volunteers with unknown studies, this study showed practically no neutralizing antibody status, received serologic evidence of infection. virus-free tissue-culture maintenance fluid. Studies M 2/14/55 and M 2/28/55-Conjunc- This method did not produce substan- tival Swabbing of Adenovirus Types 3 and 4 tial evidence of infection as only one When the conjunctival component of volunteer who received virus (Group 1) naturally acquired illness from Type 3 showed a rise in complement-fixing anti- adenovirus had been established,13 bodies. Two to five days after inocula- another study was undertaken at the tion, mild respiratory illness chiefly Maryland Reformatory, using the con- manifest by nasal obstruction and dis- junctival route of inoculation. As pre- charge occurred with near equal fre- viously reported 10 40 volunteers had quency in all study groups, including adenovirus (20 received Type 3 and 20, the control group. It tended to be less Type 4), grown in monkey kidney cells, frequent and less severe than in the swabbed on the palpebral conjunctiva former study as no volunteers were suf- of one eye and virus-free tissue-culture ADENOVIRUSES VOL. 46 1137 maintenance fluid swabbed on the con- adenovirus Types 1 and 5, also grown junctiva of the other (control) eye, in monkey kidney cells, would produce without the volunteer knowing which illness similar to that produced by Types eye received virus. Twenty volunteers 3 and 4 virus in the previous study; (b) were without detectable preinoculation if Type 4 virus grown in HeLa cells homologous neutralizing antibodies, and would produce illness to the same extent in two to seven days each developed as that grown in monkey kidney cells; definite conjunctivitis in the eye and (c) if Type 4 virus grown in swabbed with virus. Virus was isolated monkey kidney cells would produce ill- from the inoculated eye in each of the ness when swabbed only on the oro- 20 volunteers, and all 20 showed a four- pharynx. A total of 91 volunteers were fold neutralizing antibody response in distributed at random into five study their convalescent serums. In contrast, groups. Each volunteer was swabbed only six of the other 20 volunteers, who on the lower palpebral conjunctiva of had detectable neutralizing antibodies each eye and on the posterior oro- in their preinoculation serums, devel- pharynx with either live virus or virus- oped conjunctivitis. Conjunctivitis oc- free maintenance fluid. Virus was curred in the eye inoculated with swabbed onto one eye and the throat virus-free inoculum in only two of the of volunteers in study Groups I and 40 volunteers, and these two instances II, onto one eye only in study Groups were secondary to conjunctivitis in the III and IV, and onto the throat only in eye receiving virus. It was concluded study Group V. During the observation that swabbing Types 3 and 4 adenovirus period neither the observers nor recipi- on the conjunctiva of susceptible volun- ents knew which site received virus. teers produced definite easily recogniz- Specimens were collected from each eye able illness resembling pharyngocon- and the throat from 78 volunteers on junctival fever. the fifth, and from all volunteers on the ninth postinoculation day and cultured Study C-3/55-Conjunctival and/or Throat for virus isolation. Swabbing of Adenovirus Types 1, 4, and 5 The inoculums produced infection as These results prompted another study indicated by postinoculation comple- at Chillicothe to determine (a) if ment-fixing antibody responses to Table 5-Study C-3/55-Number of Volunteers with Complement-Fixing Antibody Response According to Preinoculation Neutralizing Antibody Titer and Postinoculation Virus Isolation and Conjunctivitis Preinoculation Postinoculation Virus Isolation Conjunctivitis Homologous N. Ab. Titer 5th or 9th Day < 1:4 1:4 > 1:4 Ques- + - Def. tionable No Totals Complement 24 fold 28 13 4 36 9 38 5 2 45 Fixing 2 fold 5 5 6 *7 9 8 3 5 16 Response None 5 15 10 t7 23 9 6 15 30 'ICr _ 1 enet 63 % A1 50 41 55 14 22 91 Per cent cnIa Per Response > 4 fold 74 39 20 72 22 69 36 9 50 *Two Type 4's showed homologous N. Ab. rise; two Type 5's and one Type I showed no such rise. t Four Type 4's showed homologous N. Ab. rise; three Type 5's showed no such rise. 1138 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH Table 6-Study C-3/55-Number of Volunteers with Fifth or Ninth Day Virus Isolation by Study Group According to Preinoculation Neutralizing Antibody Titer (Number with Virus Isolation/Number of Volunteers) Throat Site Inoculated Eye and Throat Eye Only Only 1 5 4 4 4 Total Virus Type Inoc.* (MK) (MK) Total (MK) (HeLa) Total (MK) I-V Per Per Per Study Group I II No. cent III IV No. cent V No. cent Preinoculation <1:4 6/9 7/7 13/16 81 8/8 8/8 16/16 100 0/6 29/38 76 Homologous Neutralizing 1:4 3/6 3/5 6/11 54 4/6 5/8 9/14 64 0/8 15/33 45 Antibody Titer > 1:4 0/1 5/11 5/12 42 0/1 1/3 1/4 25 0/4 6/20 30 Total 9/16 15/23 24/39 62 12/15 14/19 26/34 76 0/18 50/91 55 * MK indicates virus grown in monkey kidney tissue-culture cells. HeLa indicates virus grown in HeLa tissue- culture cells. homologous or heterologous Type 2 pharynx, and only four of these had a antigen and by successful virus recov- complement-fixing antibody response. It eries on the fifth or ninth postinocula- is noteworthy that 29 (91 per cent) of tion day when virus would be expected 32 susceptible volunteers were infected only if proliferation had occurred. by swabbing Types 1, 4, or 5 virus on Table 5 shows significant correlation their palpebral conjunctiva. between the complement-fixing antibody Table 7 shows the percentage of vol- response and (a) susceptibility as indi- unteers with various clinical mani- cated by no detectable preinoculation festations according to preinoculation neutralizing antibodies, (b) infection as neutralizing antibody status and to post- indicated by virus recovery, and (c) inoculation virus isolation on the fifth illness as indicated by the occurrence of or ninth day. In general, the frequency conjunctivitis. As shown in Tables 6 of each manifestation in volunteers and 7 virus recovery was significantly without detectable neutralizing anti- correlated with various indexes of sus- bodies was higher than in those with ceptibility and illness. such antibodies. Also the frequency in Table 6 shows that infection as indi- volunteers who shed virus was higher cated by postinoculation virus recovery than in those from whom virus was not varied inversely with increasing prein- recovered. Though not shown in the oculation neutralizing antibody titer. table, the frequency of illness manifes- Postinoculation virus recovery occurred tations was higher in volunteers with a to a nearly equal extent in each of study complement-fixing antibody response Groups I-IV. Not shown in the table, than in those without. Thus all the volunteers with infection as indicated manifestations listed are considered to by a complement-fixing antibody re- be associated to a greater or lesser ex- sponse had a similar distribution in tent with the virus received. Since con- Groups I-IV. No virus was recovered junctivitis was the most definitive mani- from the 18 volunteers in Group V who festation, was the most frequent in received Type 4 virus only on the oro- occurrence, and was strikingly associated ADENOVIRUSES VOL. 46 1139 with both the absence of prechallenge less severe than in the eye receiving neutralizing antibodies and virus recov- virus. ery, and since all other manifestations Table 8 shows the occurrence of con- occurred more commonly in persons junctivitis in volunteers by study group with conjunctivitis than in those with- according to preinoculation homologous out, conjunctivitis was used as an index neutralizing antibody titer and postin- of illness. oculation virus isolation on the fifth or Additional evidence that the inocu- ninth day. As with infection, the fre- lated viruses induced illness was found quency of conjunctivitis varied inversely through comparing local manifestations with increasing titer of neutralizing at the site of virus inoculation and at antibodies and was uniform in each of the site of inoculation of the virus-free study Groups I-IV. No conjunctivitis maintenance fluid. Using the paired eye occurred in volunteers in Group V who as a control minimizes the influence of received virus only on the oropharynx; psychologic, irritant, or allergic factors however, six of the 18 developed naso- which may exist when different indi- pharyngeal catarrh. Conjunctivitis was viduals are used as controls. Fifty-five more frequent in volunteers from whom of 73 volunteers inoculated with virus virus was recovered. onto the conjunctiva of one eye devel- Clinically the conjunctivitis was non- oped definite conjunctivitis in that eye. purulent and follicular, frequently in- Only five persons developed conjunc- volved both the palpebral and bulbar tivitis in the eye which received virus- conjunctiva, and was often accompanied free inoculum. Each of these five had by blepharospasm. It was first notice- bilateral conjunctivitis, which was able from three to seven days after probably due to a secondary spread, inoculation, lasted from two to ten days, since the finding occurred in the control and was indistinguishable from that eye at a later date and was definitely previously described for naturally oc- Table 7-Study C-3/55-Percentage of Volunteers Having Various Indexes of Illness According to Preinoculation Neutralizing Antibody Titer and According to Postinoculation Virus Isolation Postinoculation Virus Preinoculation Homologous Isolation Totals N. Ab. Titer (5th or 9th Day) Per Number of Illness Indexes < 1:4 1:4 > 1:4 Yes No cent Volunteers Investigators' Opinions 63 33 20 68 15 44 40 Subjective Symptoms 76 52 30 72 39 57 52 Headache 32 3 10 22 10 16 15 Cough 26 33 0 28 15 22 20 Nasal Discharge or Obstruction 50 36 20 44 32 38 18 Sore Throat 24 15 20 30 7 20 18 Eye Complaints 37 21 5 44 0 24 22 Objective Findings 79 61 35 88 32 63 57 Throat Signs 21 15 5 24 5 26 14 Conjunctivitis 79 58 30 88 27 60 55 Number of Volunteers 38 33 20 50 41 91 1140 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH Table 8-Study C-3/55-Number of Volunteers Having Conjunctivitis by Study Group According to Preinoculation Neutralizing Antibody Titer and According to Virus Isolation on Fifth or Ninth Day (Number with Conjunctivitis/Number of Volunteers) Throat Site Inoculation Eye and Throat Eye Only Only 1 5 4 4 4 Total Virus Type Inoc.* (MK) (MK) Total (MK) (HeLa) Total (MK) I-V Per Per Per Study Group I II No. cent III IV No. cent V No. cent Preinoculation <1:4 7/9 7/7 14/16 87 8/8 8/8 16/16 100 0/6 30/38 79 Homologous Neutralizing 1:4 5/6 3/5 8/11 73 4/6 7/8 11/14 78 0/8 19/33 58 Antibody Titer > 1:4 0/1 5/11 5/12 42 0/1 1/3 1/4 25 0/4 6/20 30 Total 12/16 15/23 27/39 69 12/15 16/19 28/34 82 0/18 55/91 60 Virus Yes 8/9 12/15 20/24 83 11/12 13/14 24/26 92 0/0 44/50 88 Isolation No 4/7 3/8 7/15 47 1/3 3/5 4/8 50 0/18 11/41 27 * MK indicates virus grown in monkey kidney tissue-culture cells. HeLa indicates virus grown in HeLa tissue- culture cells. curring pharyngoconjunctival fever.13 volunteers, including those inoculated No keratitis developed in any of the only on the pharynx. volunteers. The preauricular lymph Cytological examination was made of node on the involved side was frequently smears taken from both eyes of 16 vol- enlarged. Pharyngitis developed in 26 unteers, each having conjunctivitis in (36 per cent) of the 73 volunteers who one eye. The smears were stained with received virus on the conjunctiva, but it Papanicolaou stain. There were no ap- occurred in only one of the 18 who re- preciable differences in smears taken ceived virus only on the pharynx. The from the affected and the paired unaf- posterior pharynx generally showed fected eyes, and no definite cytoplasmic vascular injection and lymph follicle or nuclear inclusions were found. Seven hypertrophy. In a few instances, acute conjunctival and 18 palatine-tonsil bi- unilateral tonsil hypertrophy with vas- opsy specimens were collected from ill cular injection and unilateral submaxil- persons.* Dr. Louis B. Thomas t re- lary lymphadenopathy occurred on the ported that histopathological examina- same side as the uniocular conjunctivi- tion of the conjunctival specimens tis. Since temperatures were taken only showed a conjunctivitis of moderate during the morning examination, fevers at other times would have been missed. * Appreciation is extended to Dr. James Twelve volunteers were hospitalized for Stanbaugh, Dr. Donald J. Lyle, and Dr. Stephen P. Hogg of Cincinnati, who visited observation and nine of these (in the Federal Reformatory, performed the Groups II-V) had temperatures over biopsies, and concurred with the clinical observations. 1000 F. Rhinorrhea and nasal obstruc- t Head, Surgical Pathology and Post tion occurred in about one-third of the Mortem Service, Pathologic Anatomy Branch, National Cancer Institute. ADENOVIRUSES VOL. 46 1141 intensity characterized predominantly assuring that each group received the by migration of lymphocytes into the same quantity of virus, it seems prob- submucosal connective tissue. Examina- able that the drops placed a greater tion of the tonsil specimens indicated quantity of virus in the conjunctival sac that the clinical enlargement was due to than the swabs. It was concluded that hypertrophy and hyperplasia of the the irritation of swabbing enhanced oc- tonsillar lymphoid tissue augmented to currence of conjunctivitis. By analogy, a slight extent by congestion of vessels conjunctival irritation from swimming and edema of the peritonsillar connec- may increase the frequency of conjunc- tive tissue. tivitis in naturally acquired infection This study, C-3/55, and the earlier and may offer a vulnerable route for Maryland study M-2/55, showed that virus invasion, thus abetting the likeli- adenovirus Types 1, 3, 4, and 5 grown hood of epidemics of pharyngoconjunc- in monkey kidney cells and Type 4 virus tival fever. grown in HeLa cells produced conjunc- tivitis and infection when swabbed on Studies C and M, 4/55-Intramuscular Inocula- the conjunctiva. No conjunctivitis oc- tion of Inactivated Vaccine and Subsequent curred when Type 4 virus grown in Challenge with Adenovirus Type 3 monkey kidney cells was swabbed only The procedures wherein a readily on the oropharynx. recognizable conjunctivitis was con- sistently induced in some 90 per cent of Study M-9/55-Conjunctival Swabbing or susceptible volunteers presented a Instillation into Conjunctival Fornix of method for evaluating an adenovirus Adenovirus Type 4 vaccine. Heat- and formaldehyde- The data on naturally occurring inactivated virus vaccines were prepared pharyngoconjunctival fever 13 indicated and their prophylactic value studied.1" that swimming may have enhanced the Such vaccines were prepared from Type occurrence of conjunctivitis. Since in- 3 virus suspensions and given intramus- fection and conjunctivitis resulted from cularly * in one or three doses to volun- swabbing various types of adenovirus teers with no detectable neutralizing on the lower palpebral conjunctiva, antibodies against Type 3 virus. No another study was set up to determine untoward reaction of any kind occurred. whether dropping the virus into the Thirty-five out of 45 vaccinated volun- conjunctival fornix would similarly teers developed neutralizing antibodies. produce conjunctivitis. Nine volunteers These 35, together with 17 volunteers had Type 4 virus swabbed on the con- having naturally acquired antibodies junctiva of one eye, and virus-free main- and 31 volunteers having no detectable tenance fluid dropped into the other eye. antibodies, were challenged by swab- Fourteen volunteers had an aliquot of bing Type 3 virus on their conjunctiva. the same virus dropped into one eye On the seventh day after challenge. and the control material swabbed onto virus was isolated from 77 per cent of the conjunctiva of the other eye. None volunteers without prior antibody and of these 23 participants had detectable from 29 per cent and 35 per cent of the preinoculation neutralizing antibodies two groups with vaccine-induced and against Type 4 virus. Eight of the nine swabbed with virus developed conjunc- * One-tenth ml of vaccine also was given tivitis, but only three of the 14 receiv- intradermally as a skin test together with a ing virus drops developed conjunctivitis. control injection of maintenance fluid. Local No conjunctivitis occurred in control edema of > 5 mm occurred 24-48 hours after eyes. Although there was no way of injection, predominantly in persons with pre- existing neutralizing antibodies. 1142 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH Figure 1-Percentage of Volunteers Developing Illness Following Type 3 APC Virus Challenge According to Prevaccine and Prechallenge Neutralizing Antibody (N. Ab. Titers) Prevoccine Prechollenge No. Percentage of Volunteers Developing Illness NAb. Titer NAb. Titer Vol. 0 20 40 60 80 100 NO VACCINE 1:8 21 I .* *.* * * * * * * * * * * *.::::::::.:.:.:. :. II c- 1:8 e 1:8 10 0 l ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ....... .. -. NO VACCINE 1 :8 | 17 7 11:8 35 r .. I'll' [ 'Definite"llLness "Questionoble" /llness No Illness (Reprinted in Essence-J.A.M.A. 159:988 (Nov. 5), 1955.) naturally acquired antibodies. Further- disease to the same extent as volunteers more, 90 per cent of volunteers without with naturally acquired antibodies. The prechallenge antibodies developed con- percentage of volunteers in each group junctivitis, as contrasted with 29 per developing definite and questionable ill- cent and 24 per cent of the two groups ness is shown in Figure 1. with vaccine-induced and naturally ac- Table 9 summarizes the occurrence quired antibodies. It was clearly evi- of conjunctivitis in 205 volunteers who dent that volunteers with vaccine-in- have had one eye swabbed with adeno- duced antibodies were protected against virus and the other eye similarly the artificially induced infection and swabbed with virus-free maintenance Table 9-Summary of Conjunctivitis Occurrence in Volunteers Having the Con- junctiva of One Eye Swabbed with Virus-Free Maintenance Fluid and the Other Eye Similarly Swabbed with Various Types of Adenovirus According to Preinoculation Neutralizing Antibody Titer (Number of Eyes Developing Conjunctivitis/Number of Eyes Swabbed) Preinoculation Homologous One Eye Swabbed with Other Eye Swabbed with Adenovirus Neutralizing Virus-Free Mainte- Types Antibody nance Fluid Titer * (Secondary Spread) Total 1 3 4 5 < 1:4 HeLa or < 1:8 Monkey Kidney Cell Test 8/92 86/92 7/9 40/43 32/33 7/7 Per cent (93) > 1:4 HeLa or > 1:8 Monkey Kidney Cell Test 2/113 45/113 5/7 17/60 15/30 8/16 Per cent (40) Total 10/205 131/205 12/16 57/103 47/63 15/23 * Monkey kidney cell test more sensitive for antibody determination at low dilutions.18 ADENOVIRUSES VOL. 46 1143 fluid. It shows that in the absence of groups depends basically upon the detectable neutralizing antibodies at the amount of respiratory illness caused by 1:4 (or 1:8 dilution in monkey kidney adenovirus and on the duration of vac- cells), 86 (93 per cent) of 92 virus- cine protection. This should be studied inoculated eyes developed definite con- particularly in children who have a high junctivitis. In contrast, conjunctivitis incidence of acute febrile respiratory developed in 45 (40 per cent) of 113 illness. Nursery studies now in progress volunteers with detectable antibodies at indicate a high prevalence of adenovirus this low level. This titer level appears infection. Nine children less than three to indicate the level of 90 per cent sus- years of age have been given the Type 3 ceptibility to a challenge-induced illness vaccine. Seven had no detectable pre- which was the chief concern in these vaccine Type 3 neutralizing antibodies studies. The serums were not titered and all seven developed such antibodies high enough to establish the antibody in their postvaccine serums. Although level necessary for a high degree of im- further studies are indicated, this sug- munity. In 10 instances conjunctivitis gests that the vaccine served as a in control eyes followed that in contra- primary antigenic stimulus and may lateral, virus-inoculated eyes, and these have prophylactic value in children. instances were considered secondary in- A live virus vaccine also deserves fections. Adenovirus Types 1, 3, 4, and exploration since the studies reported 5, all that have been tested to date, pro- here indicate that nasopharyngeal inocu- duced conjunctivitis in similar fre- lation of adenovirus resulted in anti- quency, nature and intensity. body formation without illness which could definitely be attributed to the Discussion viruses used. In addition, live adeno- viruses have been inoculated in large Swabbing the conjunctiva with adeno- quantities intra-arterially and into virus consistently produced laboratory malignant tumors of persons without evidence of infection and produced a apparent ill effect.'5 Live virus vac- readily recognized clinical illness in 94 cines, however, present a problem in per cent of volunteers when they were assuring absence of other live virus con- susceptible as indicated by absence of taminants which may be dangerous.'6' 17 neutralizing antibodies. An inactivated The nasopharyngeal route of adeno- Type 3 adenovirus vaccine was prepared virus inoculation did not produce con- which artificially induced antibody for- j unctivitis; however, nasopharyngeal mation and was accompanied by a con- catarrh characterized by rhinorrhea, version from susceptibility to immunity. nasal obstruction, and throat discomfort The demonstration that a monovalent occurred 2-4 days following inoculation vaccine would protect against a chal- in some 30 per cent of volunteers. This lenge-induced illness stimulated the symptom complex was usually called a preparation of a polyvalent Type 3, 4, "cold" and seemed similar to many ill- and 7 adenovirus vaccine. Field studies nesses occurring in volunteer studies re- are under way to determine whether this ported by others.2 12, 18, 19 In the vaccine will protect against naturally present studies, these "colds" tended to occurring adenovirus disease. occur more frequently in volunteers A suitable vaccine would appear to without homologous neutralizing anti- be especially useful in military recruits bodies and in volunteers who had a who are reported to have a high inci- complement-fixing antibody response, dence of disease due to adenovirus.14 but the differences were not statistically The value of such a vaccine in other significant and the "colds" could not 1144 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH definitely be attributed to the viruses "colds." The possible influence of irri- inoculated. In addition, similar "colds" tant, psychologic, allergic, and other fac- occurred within 2-4 days in a nearly tors deserves further study. equal proportion (some 30 per cent) of The "colds" presumably due to control volunteers who either were not viruses may be caused by infection with inoculated or received virus-free main- agents which do not produce a lasting tenance fluid. This occurred in each of immunity or they may be due to many the three studies where such controls immunologically distinct viral agents were observed simultaneously with which separately induce sustained im- virus-inoculated groups. The time of munity. In the spectrum of respiratory occurrence of these "colds" did not sug- disease due to various known viral gest secondary spread from the virus- agents (influenza, psittacosis, adeno- inoculated volunteers and none showed viruses, etc.) the milder illnesses are a complement-fixing antibody response. often interpreted as "colds." These mild In the two early Maryland studies the reactions may be due to constitutional volunteers were observed under group factors of the host, to route of invasion, hospital isolation and in Study C 3/54 to virulence, or to dosage of the agent. they were not generally isolated. There On the other hand, some "colds" may was no evidence of a concurrent epi- represent an "immediate" or "accel- demic in the nonstudy population of erated" reaction to reinfection with either institution during the periods homologous virus in partially immune when these studies were carried out. or sensitized individuals. Another These findings indicate that a sub- sibility is that some colds may be pos- due stantial number of the "colds" observed to reactivation of latent viral infections in these volunteer studies were not such as herpes or dormant adenoviruses. caused by the inoculated viruses. In Volunteer studies provide limited but view of these observations, it becomes valuable information; however, one pertinent to reexamine hypotheses on should be wary of extrapolation of re- the etiology of "colds." The general sults to the phenomenon of the natural epidemiologic picture is compatible occurrence of "colds." Volunteers are with a view that "colds" are caused by not representative of the general popula- viruses. Although some "colds" may tion; the method, dosage, and route of be due to viruses, others may be due to inoculation are artificial; the inoculums other causes. It is of interest that the are complex substances; the susceptibil- Army studies 2 have shown no lasting ity-immunity status is generally not immunity to "colds" when individuals known and may vary tremendously in were rechallenged with aliquots of the small groups; and the clinical manifes- same inoculums. This may be inter- tations are difficult to measure with preted to indicate that either these par- finite accuracy. In spite of these limi- ticular "colds" were not due to viral tations, the present report exemplifies agents or that the presumed viral agents that volunteer studies, when properly did not produce the lasting immunity planned, can answer specific questions so common in other viral infections. In that otherwise might require years of the present studies, "colds" developed observation under natural circum- in volunteers who were not inoculated stances. and in those inoculated with virus-free maintenance fluid. In one study, seven volunteers were noted to complain of Summary and Conclusions nasal irritation several hours after Studies of adenovirus infection, im- inoculation and all seven later developed munity, and disease in adult volunteers ADENOVIRUSES VOL. 46 1145 have been described. The intranasal in- bodies, and no untoward reactions oc- stillation of adenovirus Types 1, 2, 3, 4, curred. Such vaccinated persons were 5, or 6 and the swabbing of the oro- protected against challenge-induced ill- pharynx with Type 4 virus have pro- ness apparently to the same extent as duced infection as demonstrated by a adult volunteers with naturally acquired complement-fixing antibody response. antibodies. Such inoculations have commonly been followed by minor respiratory illness, ACKNOWLEDGMENT-The inmates and staff of the Federal Industrial Reformatory, Chilli- chiefly manifest by an afebrile naso- cothe, Ohio, and the Maryland State Reforma- pharyngeal catarrh, which could not be tory for Males, Breathedsville, Md., cooperated attributed to infection with these viruses. in this study. We are greatly indebted to Both infection and illness were readily Warden R. P. Hagerman, M.D., and Chief Medical Officers Edward Slaten, M.D., and produced in susceptible volunteers by T. P. Hackett, M.D., of the Chillicothe Re- swabbing the lower palpebral conjunc- formatory, and to R. L. Clopper, assistant tiva with adenovirus Types 1, 3, 4, or 5. superintendent of the Maryland Reformatory, The frequencies of infection and illness who actively assisted in the performance of were similar when virus grown in either these studies. HeLa or monkey kidney cells was used. REFERENCES 1. Enders, 3. F.; Bell, J. A.; Dingle, J. H.; Francis, Infection was demonstrated by recovery T., Jr.; Hilleman, M. R.; Huebner, R. J.; and of the homologous virus from the eye Payne, A. M. N. "Adenoviruses": Proposed Group Name for the New Respiratory-Tract Viruses (AD. or throat from five to nine days after RI-APC-ARD Agents). Science 124, 3212:119-120 inoculation and by complement-fixing (July 20), 1956. 2. Commission on Acute Respiratory Diseases. Experi- antibody response. Illnesses occurred mental Transmission of Minor Respiratory Illness to from two to seven days following inocu- Human Volunteers by Filter-Passing Agents. I. Demonstration of Two Types of Illness Characterized lation, generally persisted from four to by Long and Short Incubation Periods and Different eight days, were occasionally febrile, Clinical Features. II. Immunity on Reinoculation with Agents from the Two Types of Minor Respira- and were characterized chiefly by a fol- tory Illness and from Primary Atypical Pneumonia. licular conjunctivitis. Often pharyngitis J. Clin. Investigation 26:957-982, 1947. 3. Rowe, W. P.; Huebner, R. J.; Gilmore, L. K.; with vascular injection and lymph fol- Parrott, R. H.; and Ward, T. G. Isolation of a licle hypertrophy was seen. These ill- Cytopathogenic Agent from Human Adenoids Under. going Spontaneous Degeneration in Tissue Culture. nesses included complaints of eye Proc. Soc. Exper. Biol. & Med. 84 570-573, 1953. 4. Huebner, R. J.; Rowe, W. P.; Ward, T. G.; irritation and discharge, nasal discharge Parrott, R. H.; and Bell, J. A. Adenoidal- and obstruction, sore throat, and occa- Pharyngeal-Conjunctival Agents. A Newly Recog- nized Group of Common Viruses of the Respiratory sionally cough and headache. Both ob- System. New England J. Med. 251:1077-1086 (Dec. jective and subjective symptoms were 30), 1954. 5. Rowe, W. P.; Huebner, R. J.; Hartley, J. W.; associated with demonstrated infection, Ward, T. G.; and Parrott, R. H. Studies of the and illness occurred predominantly in Adenoidal-Pharyngeal-Conjunctival (APC) Group of Viruses. Am. J. Hyg. 61:197-218 (Mar.), 1955. volunteers without detectable prechal- 6. Rowe, W. P.; Hartley, J. W.; and Huebner, R. J. lenge homologous neutralizing anti- Additional Serotypes of the APC Virus Group. Proc. Soc. Exper. Biol. & Med. (In press.) bodies. Swabbing viruses onto the con- 7. Jawetz, E.; Kimura, S.; Tricholas, A. N.; Thygeson, junctiva produced a higher frequency P.; and Hanna, L. New Type of APC Virus from Epidemic Keratoconjunctivitis. Science 122:1190- of conjunctivitis than did dropping virus 1191, 1955. 8. Hilleman, M. R., and Werner, J. H. Recovery of into the conjunctival fornix. New Agent from Patients with Acute Respiratory Heat- and formaldehyde-inactivated Illness. Proc. Soc. Exper. Biol. & Med. 85:183-188, 1954. virus vaccines were prepared against 9. Ginsberg, H. S.; Badger, 0. F.; Dingle, J. H.; Type 3 adenovirus. When adult volun- Jordan, W. A., Jr.; and Katz, S. Etiologic Rela. tionship of the RI-67 Agent to Acute Respiratory teers without detectable Type 3 neutral- Diseases (ARD). J. Clin. Investigation 34:820 izing antibodies were inoculated intra- (June), 1955. 10. Ward, T. G.; Huebner, R. J.; Rowe, W. P.; Ryan, muscularly with these vaccines, 78 per R. W.; and Bell, J. A. Production of Pharyngocon. cent developed Type 3 neutralizing anti- junctival Fever in Human Volunteers Inoculated with APC Virus. Science 122:1086-1087, 1955. 1146 SEPTEMBER 1956 AMERICAN JOURNAL OF PUBLIC HEALTH 11. Huebner, R. J.; Bell, J. A.; Rowe, W. P.; Ward, Respiratory Disease Virus RI-67. A.J.P.H. 45, 203- T. G.; Suskind, R. G.; Hartley, J. W.; and 210, 1955. Paffenbarger, R. S., Jr. Studies of Adenoidal- 15. Smith, R. R.; Huebner, R. J.; Rowe, W. P.; Pharyngeal-Conjunctival Vaccines in Volunteers. Schatten, W. E.; and Thomas, L. B. Studies on the J.A.M.A. 159 986-989, 1955. Use of Viruses in Treatment of Carcinoma of the 12. Ward, T. G., and Procter, D. F. Isolation of a Cervix. (To be published.) Common Cold Virus in Chick Embryos and the 16. Hull, R. N. Personal communication. Clinical Manifestations It Produces in Human 17. Rowe, W. P.; Huebner, R. J.; Bell, J. A.; and Volunteers. Am. J. Hyg. 52 91-106, 1950. Hartley, J. W. (Unpublished data.) 13. Bell, J. A.; Rowe, W. P.; Engler, J. I.; Parrott, 18. Topping, N. H., and Atlas, L. T. A Note Regarding R. H.; and Huebner, R. J. Pharyngoconjunctival Isolation of an Agent. Science 106:636-637, 1947. Fever-Epidemiological Studies of a Recently Recog. 19. Andrewes, C. H. Adventures Among Viruses. III. nized Disease Entity. J.A.M.A. 157:1083-1092, 1955. The Puzzle of the Common Cold. New England J. 14. Hilleman, M. R.; Werner, J. H.; Dascomb, H. R.; Med. 242:235-240, 1950. Butler, R. L. Epidemiologic Investigations with World Multiple Sclerosis Literature Through a grant of the National Multiple Sclerosis Society to the Excerpta Medica Foundation of Amsterdam, Holland, abstract of the world's current litera- ture on demyelinating diseases will be available in the English language, reportedly for the first time. The results will be published in a monthly journal containing 100-150 abstracts and distributed among scientists and physicians, universities, and libraries throughout the world. It is hoped that by bringing to scientists a greater awareness of the world's literature on the disease, achieving the control of multiple sclerosis will be hastened. Frederick L. Stone, M.D., medical director of the National Society, is a member of the 10-man editorial board of scientists that will oversee the work. Seven others are drawn from as many United States medical schools and two are Europeans.