Report of Meeting of THE COMMISSION ON NEUROTROFIC VIRUS DISEASES of the u. s. Army Epidemiological Board »l I held at The Rockefeller Institute for Medical Research New York City 18 October 1945 Owing to the limited time available at the meeting some of the reports listed in these minutes were "read by title". They are marked with an asterisk. The meeting was called to order at 10:00 AM at the Rockefeller Institute. The following were present: Members of. the. Commission.- Drs. C. D. Aring, G. D. Gammon, W. McD. Hammon, Maj. W. P. Havens, Jr., Dr. C. A. Janeway, Lt. Col. A. B. Sabin, Capt. R. W. Schlesinger, Dr. R. Ward, and Dr. J. R. Paul, Director. nAssociate Members”.- Drs. P. K. Olitsky and J. Casals. Representing the Surgeon Generalfs Office.- Lt. Col. Aims C. McGuinness. Representing the Army Medical School." Col. H. Plots, Lt. Col. J. E. Smadel, Lt. J. Warren, Maj. L. Whitman. Guests.- Drs. D. M. Horstmann, H. A. Howe, K. F. Maxcy, J. L. Melnick, Mr. J. T. Riordan, Maj. E. B. Schoenbach, Dr. M. Theiler. TABLE OF CONTENTS Page Introduction and Future Program by Dr* Paul and Col. McGuinness 1 1. Encephalitis Outbreak on Okinawa by Col. Sabin 1 2* Okinawa Encephalitis Investigations by Dr* Hammon 6 3* Preparation of Japanese B Encephalitis Vaccines from the Infected Chick Embryo by Lt. Warren 9 4. Diphtheritic Polyneuritis by Dr. Gammon and Maj. Schoenbach 11 5. Vaccination of Human Beings with Dengue Virus Modified by Passage in Mice by Col. Sabin and Capt. Schlesinger 15 6. Dengue Virus in Chick Embryos by Capt. Schlesinger and Col. Sabin 16 7. Dengue Neutralization Tests in Mice by Capt. Schlesinger and Col. Sabin 18 8. Report of Committee on Neutralization Tests by Dr* Olitsky 18 9* *The Current State of Production of Equine Encephalomyelitis Virus Vaccines by Dr. Olitsky 19 10. ♦Russian Spring-Summer Encephalitis. Final Report on Vaccination Studies by Drs. Casals and Olitsky 19 11. ♦Effect of Normal Serum on Titer of Virus by Drs. Casals and Olitsky 20 12. ♦Diagnosis of Encephalitis Viruses by Animal Inoculation and Complement- Fixation Tests by Dr. Casals 20 13. Experiments in Infectious Hepatitis by Maj* Havens 21 14. Serum Jaundice by Dr. Janeway 25 15. Infectious Hepatitis Lesions in Livers of Normal Mice by Drs. Olitsky and Casals 27 16. Polionyelitis in 1945. The Outbreak at Fort McClellan, Alabama. by Drs. Ward and Paul 28 17* Civilian Epidemics of Poliomyelitis in the East—1945 by Dr. Paul 30 18. Encephalitis and Poliomyelitis in the West—1945 by Dr* Hammon 30 19. Murine Poliomyelitis Virus in the Chick Embryo by Mr. Hi or dan 38 20. Ply Abatement Studies in Urban Epidemics of Poliomyelitis by Drs. Melnick and Ward 39 INTRODUCTION, by Dr. J. R. Paul. In thanking .members and guests for coming. Doctor Paul stated that this might be the last formal meeting of the Commission. A brief account of some of the accomplishments of the group during the past three years was reviewed, with an expression of appreciation and pride in the Commission's work during the war years. FUTURE PROGRAM, by Lt. Col. A. C. McGuinness, M.C. Colonel McGuinness spoko briefly on postwar plans, stating that the Army Epidemiological Board would eventually become a part of the recently organized Any Medical Department Research and Development Board* Present plans for the postwar Army Epidemiological Board call for a Central Board composed of five members, and in addition about twenty Consultants. There will be no permanently organized Commissions as at present. Commissions will be constituted from time to time for investigation of special diseases and epidemiological problems, and membership on Commissions will change from time to time as need may indicate. While a budget for contracts with universities is contemplated, nothing definite has been done about this as yet, and he advised that we should not count on having Any funds after 1 July 1946. 1. ENCEPHALITIS OUTBREAK ON OKINAWA by Lt. Col* A. B. Sabin, M.C. History of Outbreak.- The first known civilian cases of encephalitis had their onset about 1 July 1945, and were detected between 7 July and 9 July, both on the mainland of Okinawa and on the adjacent island of Heanza. By the end of July at least 80 cases had been found among civilians of whom 22 died, but only 3 suspected and 3 probable cases (only one of which has been confirmed by serological tests subsequently) among American military personnel* On the isolated islands of Heanza and Hamahika the epidemic apparently terminated by the end of July with an incidence of 4.7 per 1,000 (31 cases among a total population of 6,547) and 3.3 per 1,000 (5 cases among 1500) respectively. The actual incidence of cases on the mainland of Okinawa has remained diffi- cult to determine since it has been estimated that only l/3 to l/6 of the total number of cases was detected. During the month of August an additional 35 to 40 civilian cases were found on Okinawa, and 18 to 20 probable cases with 2 deaths occurred among American military personnel. A small number of additional civilian and military oases occurred during September. The death rate among civilians was about 25 per cent despite the fact that only the moderately severe and severe cases wore detected. Approximately 80 per cent of the civilian cases (94 of 118) occurred in infants and children 15 years of ago or younger. Native physicians indi- cated that "summer encephalitis" predominantly omong the younger age groups is an annual occurrence on Okinawa usually with an incidence of 5 per 10,000, although one year was recalled in which the incidence was 17 per 10,000. Identification of Etiological Agent.- The etiological agent of the outbreak was identified as the Japanese B encephalitis virus by complement fixation and neutralization tests on convalescent sera and by isolation of a single strain of the virus from the brain of a fatal civilian case. This work was done by members of the Naval Medical Research Unit No. 2 (NAMRU No. 2) and myself. Many attempts were made to isolate virus from the blood and cerebrospinal fluid of civilian and military cases but with consistently negative results. Isolation of the virus from human brain tissue v/as also difficult since no virus could be isolated from certain cases which exhibited typical lesions. Clinical Manifestations.- The clinical manifestations corresponded in general to previous descriptions of Japanese B encephalitis and were not unlike those seen in the summer encophalitides in the U.S.A# "Eye signs" (dissociated eye movements, strabismus, diplopia, nystagmus, pupillary changes) were not at all infrequent, however, in this outbreak. Pathology,- Studios were made of the train and cord of the fatal Army case and Lt, Comdr. Zimmerman of NAMRU No* 2 examined tissue from a number of civilian cases and from one marine. The extensive involvement of the Purkinje cells in the cerebellum was perhaps the least expected finding. Otherwise the nature and distribution of the lesions wore compatible with the diagnosis of encephalitis. No such Purkinje cell lesions were found in the brains of two mice inoculated intracerebrally with the Okinawa strain of virus. Entomological Aspects.- The most prevalent mosquito in the encephalitis foci was Culex quinquefasciatus. Rare specimens of Armigeros subalbatus were taken and a few larvae, but no adults, of Aodes togoi were also found. The NAMRU No, 2 entomologists also collected some Culex bitaeniorhynohus and Aedes albopiotus on Heanza, and Culex tritaoniorhynchus on the northern end of Okinawa. Several attempts to isolate virus from pools of Culex quinque- fasoiatus yielded negative results; the more extensive tests carried out with this and other species of mosquitoes by the NAMRU No. 2 team have also yielded negative results. Search for Ectoparasites and Extrahuman Hosts,- No mites were found on the few chickens which remained on the island. Two pools of Trombidiid mites were tested by mouse inoculation—one collected for mo by Capt. Parsons, Sn.C., from a Heanza dove, and another derived from doves and other birds on Heanza collected by the NAMRU No. 2 group-~with negative results. No mites were found on the horses, goats, or pigs which were examined. A few parasitid mites (not yet identified further), not enough for a test, were found on a few rats by Capt. Parsons, as well as the NAMRU No. 2 entomologists. A number of neutralisation tests have already been completed on the sera obtained from chickens, horses, and goats, and others are in progress. Neutralizing antibodies for the Okinawa strain of Jap. B encephalitis virus have already been found in the horse and goat sera but not in the chicken sera. Lt. Horace Hodes, USNR, of NAMRU No. 2 has informed me that he has obtained positive 4 complement fixation tests with each of 5 hors© sera from Okinawa hut not with the serum of a horse from Guam, used for control. Some Epidemiologic Features.- It is perhaps noteworthy that the cases of encephalitis were limited to the northern portion of Okinawa to which almost the entire civilian population had been moved. Although less than a third of all the military personnel were in this northern area, all the encepha- litis's cases occurred among them, while not a single definite case was reported among the remaining 2/3 or more of the forces occupying southern Okinawa, which was not vaccinated and which was no more free of the prevalent mosquitoes. It almost seemed as if the reservoir of the virus had boon moved with the native population or the domestic animals which accompanied it* The outbreak followed the usual climatic aspects, i.e«. there wore heavy rains in April and May followed by at least 4 to 5 weeks of a very hot, dry spell before the first cases appeared early in July. Most of July uontinued hot and dry but ended 7dth heavy rains, while August was stated to have had more rain than usual. Vilhat we looked for, and did not find, was direct evidence for the working hypothesis that a mite-like insect, constituting the real reservoir of the virus, gives rise to viromia in some of the domestic animals, with subsequent dissemination to human beings and other hosts by the mosquitoes which breed in large numbers during the hot spell following the rainy season. This may still be the true epidemiologic cycle despite the fact that neither the "mite” nor the mosquito which might have carried this virus have as yet been found on Okinawa. Considering the unusually crowded conditions under which the natives lived, there was little to favor any concept of direct human to human spread of the encephalitis. Neutralizing Antibodies in Native Okinawans pf Different Ages«- 62 sera from individuals of 6 different age groups ranging from 1 to 61 years were collected for tests as a check on the hypothesis that Okinawa is an endemic area and that most of the adult population had already had the disease. The neutralization tests on these sera using the Okinawa strain of virus are now in progress, and one test -which has gone far enough shows the absence of antibody in at least 9 of 10 sera from the 1 to 5 age group as contrasted with the probable presence of antibody in 8 o|r 9 sera from the 31 to 51 age group. Neutralizing and Complement Fixing Antibodies in Sera of Military Personnel with a Clinical Diagnosis of Encephalitis.- These tests are still in progress but there is already an indication that no neutralizing anti- bodies are demonstrable in the convalescent sera of some of the severe as well as mild cases, clinically diagnosed as encephalitis, although they have been demonstrated in others. Vaccination Program.- Although 800 to 1,000 of the most exposed mili- tary government personnel were vaccinated between 15 and 20 July, the decision to vaccinate the military personnel stationed near civilian foci of infection was reached on 1 August at a time when the future course of the outbreak could not be predicted. It was not deemed advisable to gamble on the chance that very few cases and deaths would occur among the military personnel, or to roly entirely on mosquito control measures. Enough vaccine had boon dis- tributed to inoculate over 82,000 individuals. Because of the great mobility of units on the island and the multiple services and commands involved, the exact data, which are being collected by Major W. G. Downs, are not yet available, but it is known that at least 50,000 and probably 66,000 of the approximately 75,000 military personnel occupying the portion of Okinawa, north of Ishikawa, were vaccinated during the month of August. No serious reactions have been reported from the vaccine. There were a small number of transitory allergic manifestations characterized by asthmatic-like reactions, urticaria, or angioneurotic oedema. The reports are not all in yet but the incidence of these "allergic" manifestations may be judged by the occurrence of only one such reaction (asthmatic and urticarial) among a group of approx- imately 5,000 marines. There were also a certain number of local inflammatory and short febrile reactions. No case suggestive of a postvaccinal denrveliratjn^ encephalopathy has been reported to date. Two very mild cases of encephali- tis had their onset 2 and 6 days respectively after the first dose of vaccine- After 1 September there were 4 mild cases, whose chief neurological manifesta- tions were nuchal rigidity, varying degree of pleocytosis, and transitory reflex charges, in individuals who had their first dose of vaccine 15 to 18 days be- fore onset# If the complement fixation tests on these patients become positive in the convalescent sera it will be possible definitely to class them as mild cases of encephalitis. Otherwise the etiological diagnosis will remain equivocal. I do not think that it will be possible to arrive at any conclusion regarding the role the vaccine might have played in keeping down the number of cases of encephalitis among the military personnel, despite the fact that not a single severe or moderately severe case of encephalitis occurred in any vaccinated person. 2. OKINAWA ENCEPHALITIS INVESTIGATIONS by Dr. W. McD. Hammon. A review of Dr. Hammon1 s Okinawa survey trip with Dr. Reeves can bo given briefly. Arrival in Okinawa, 26 August. Headquarters at Kana at the Military Government Encephalitis Research Hospital for about one week during which time native cases which were hospitalized thoro were seen. Epidemiological data were reviewed and preliminary observations made on mosquito population. Mosquitoes had been practically eliminated on that section of the coast so another area was investigated at Nago with the 87th Field Hospital. These headquarters were kept until the time of leaving Okinawa on 20 September. The report of the studies undertaken which included collection of sera from military and native cases, from local animals and birds, col- lection of mosquitoes and human autopsy material has been filed with the records of the Neurotropic Virus Disease Commission as of the date of 9 October 1945. Subsequent and new data %m as follows. Mosquitoes*- Half of the Culex guinguefasoiatus collected have been tested for virus* No virus has been detected. This was not wholly unexpected as the mosquitoes were collected after the subsidence of the epidemic# It has been the experience in the Yakima Valley with Western equine encephalomyelitis and St. Louis encephalitis viruses that infected mosquitoes are found early in the summer before the outbreak in human beings and not later on. Sera of Military Cases*- A number of neutralization tests against the Jap B virus have been performed, using the least amount of virus which will £ kill all mice with fai/ regularity. Very few convalescent military oases gave a slight degree of protection, of no significance in most instances. However, when a few of these together with other Okinawa sera were tested with our chick embryo antigen for complement fixation (C.F.), it was observed that those without any demonstrable neutralizing antibody were negative and most of those with very slight protection fixed complement, 2 of thorn in titers of li32 and 1*64. Sera from 7 convalescent native cases were also tested by both methods* The undiluted serum of 5 ($74,7,65,86 and 90) showed at least some slight protection, and had C. F. titers of 1:4 to 1:S2, while tho other two were entirely negative by both tests. As a result of the complement fixation tests it is felt that the very low neutralization test titers may possibly be significant. Even with this generous interpretation only 7 of tho 22 Military "cases11 from which sera were obtained gave serological evidence of infection with Jap B virus. Two of these had been vaccinated and showed no increase in neutralizing titer between 2 specimens collected, one (Liacey), showed C.F* of questionable significance in both specimens, the other (Brogan) gave a titer of 1:16 in the 8 day serum but tho later serum gave unsatisfac- tory results with a control antigen* Sera of Vaccinated Military Personnel*- Of 16 vaccinated individuals tested for neutralizing antibodies, only 1 was positive. The latter had been stationed in an area containing many native cases, and had developed also a C.F. titre of 1 to 4* These data suggest that vaccination as performed on 8 Okinawa did not give rise to neutralizing antibodies in a high proportion of individuals. Native Cases,- Of 15 convalescent sera from native oases tested for neutralizing antibody, 7 had neutralizing antibody to a significant degree. As mentioned above, 7 that were checked hy C.F. correlated perfectly with no protection or with minimum detectable protection. No serial sera on any native cases were tested to rule out the presence of Jap B antibody in the acute phase sera. Normal Native Sera,- Some of the normal native sera of the age groups up to 20 have been tested for neutralizing antibodies. The results are as follows: AGES 10 Ho. LD50 doses neutralized 50 Pos. Neg. Pos. Neg. 0-4 0 7 0 7 5-9 1 7 0 8 10 - 14 6 1 4 3 15 * 19 5 0 2 3 It may b© tentatively concluded from these small numbers that few natives under 10 years of age have been previously infected and that in the ago group from 10 to 20 infection has occurred in a considerable proportion. Commenting on Col. Sabin1 s statement that he (Sabin) had been unable to detect virus in blood or spinal fluid of patients. Dr. Hammon suggested that many of the samples of blood and spinal fluid may have been taken from patients not having Jap B encephalitis. Col* Sabin answered that 6 of 7 cases proved serologically as Jap B encephalitis had failed to yield virus in the blood or spinal fluid. Col, Smadel asked -why complement fixing antibody titres were so high when neutralizing antibody titres wore relatively low. He raised the ques- tion of whether the subjects had also received typhus vaccine because the sera of 20% of troops receiving "booster" doses of typhus vaccine developed complement fixation with normal chick tissue. Dr. Hammon replied that no fixation was obtained with normal chick embryo used as control entigen. Col. Sabin observed that none of Hammon*s group of patients included early July cases; nevertheless even certain of the latter considered as good examples of Jap B encephalitis failed to show neutralizing antibody. 3. PREPARATION OF JAPANESE B ENCEPHALITIS VACCINES FROM THE INFECTED CHICK EMBRYO by Lt. J. Warren, Sn.C. In this the second report to be presented before the Commission on this subject, progress is described with the method of preparation of a chick embryo vaccine against Japanese B encephalitis. Preliminary tests of this vaccine in the Virus Laboratory at the Array Medical School and at the National Institute of Health, indicate its potency to be comparable to the present commercial mouse brain vaccine. Preparation of Vaccine.- The seed virus used in the inoculation of eggs was harvested and stored as a 10% suspension of infected embryo in whole filtered chicken serum. Such seed suspensions withstood storage at -70°C in sealed ampules for more than one month with no appreciable loss of titer. Both the seed virus and the material for vaccino was obtained from embryonatod eggs which were 7 or 8 days old when infected via the chorio- allantoic sac. These eggs were subsequently incubated at 35°C for throe days at which time the embryos were harvested. The whole embryo was removed from groups of 20 to 200 infoctod eggs. The lens and retinal cup wore dissected out and discarded. The remaining embryo tissue was then homogenized in a Waring blendor for 5 minutes with sufficient physiological saline solution, buffered at pH 7.4 to make a 10 or 20 per cent suspension by weight. The resulting suspension was strained through gauze. After samples were removed for sterility tests and for titration of infeotivity, sufficient formaldehyde U.S.P. was added to give a final concentration of The material was then placed in the refrigerator for two weeks, following which the vaccine was assayed for potency. All of the chick vaccines have teen assayed in the same manner as that used for commercial mouse brain vaccine, namely the mouse protection test devised by Sabin and Duffy. Results*- In order for a vaccine prepared from the embryoiated egg to merit consideration for human use it must satisfy the standard of potency accepted for a mouse tissue vaccine. This requirement states thab a vaccine may be regarded as of acceptable potency if the minimal immunogenic dose protecting 50$ of the mice (M.I.D. 50$) is 0.01 cc. or less. The lowest M.I.D. 50$ which wo cou3d obtain with a 10$ embryo vaccine -7 5 was 0*03 cc. This vaccine had an initial infective titer of 10 * . By centrifugation at high speed in the Sharpies or air-driven angle ultra- contrifugo i£ was possible to achieve a 2 to 3 fold increase in potency. How- ever this concentration of antigen was obviously still insufficient. It was then found that by increasing the concentration of infected embryo to 20$, vaccines could bo prepared which would meet the accepted requirements. Thus, 8 of 11 twenty per cent vaccines assayed between 0.002 oc. and 0.01 cc. There is some correlation between the titer of virus in the vaccine before -.7 c. inactivation and its potency. In general, infective titers of 10 or greater are required for a successful vaccine. Attempts to concentrate the chick embryo vaccine on calcium phosphate or potassium alum met with failure. On the other hand the potency of these preparations could be increased two or three-fold by the use of an adjuvant consisting of equal parts "Falba" and mineral oil. In these experiments a 2$ suspension of adjuvant in physiological saline was used as the diluent in making the vaccine assay. Because ”Palba,f cannot be used in tho vaccination of man we are at present testing related but less harmful compounds, for any similar adjuvant activity. No tests of these embryo vaccines have as yot been performed in man. Discussion*- Col. Sabin mentioned 2 factors to be considered in deciding what type of vaccine should be prepared for the summer of 1946* 1#) Commercial houses are already making mouse brain vaccines. 2.) How desirable is it to give another chick embryo vaccine to individuals already vaccinated with chick material? Col. Smadel observed that the response to chick embryo Jap B vaccine in human beings is unknown. Col. Plotz stated that the amount of egg white in the chick embryo was much less than that in the yolk sac. He reported that there Iiad been 14 serious reactions to chick embryo vaccines and some deaths. 4. DIPHTHERITIC POLYNEURITIS by Dr. G.D. Gammon and Maj. E.B. Schoohbach, M. C. A group of 70 cases of multiple peripheral neuritis among German Priso- ners of War was studied. The chief problem was to determine what proportion were post-diphtheritic. A careful history was taken including the German laboratory data and treatment, and a comprehensive neurological examination made. From this it was found that the cases fell into several groups which had characteristic clinical syndromes; a few did not fit into these definite categories. The division was as follows; 1) Diphtheritic neuritis, 42; 2) sulfonamide neuritis, 13; 3) post-infectious neuritis from wound infection with severe gonoral sepsis, 6; and 4) unclassified cases, 9. All of these, and a group of control cases, were cultured on two suc- cessive days , for C. diphtheriae from nose and throat, and serum antitoxin levels were determined by the method of Fraser. The controls consisted of 112 individuals without diphtheria and 17 cases of recent diphtheria. The total group comprised 199 individuals. The outstanding finding was that 17$ of the normal controls carried virulent diphtheriae. This exceeded, though not significantly, the posi- tives in the diphtheritic neuritic group. Between the various neuritides there was no- significant difference in the percentage of positives. This baoteriologic study, therefore, did not differentiate diphtheritic neuritis from other types. Antitoxin levels in serum were lower in the cases of recovering diphtheria and in the diphtheritic neuritis cases than in the control group. But between the different neuritides there was no significant difference. This test, therefore, also failed to distinguish diphtheritic neuritis from the other types. The failure of the culture method to differentiate is probably due to two causes: 1) the very high carrier rate and, 2) the fact that the neuritis cases studied late at a time ■when organisms were disappearing from the nose and throat and the group was approaching the positivity of the sur- rounding community. In both tests the small size of the non-diphtheritic groups militated against differentiation. Despite these negative results, on clinical grounds, the various types were considered distinctive. Thus, post-diphtheritic neuritis was character- ized by l) slow development of the syndrome occurring 4 to 6 weeks after onset of diphtheria, and 2) paralysis of palate and pupil accommodation. Sulfonamide (methyl sulfathiazole or "Yleron”) neuritis was characterized by sudden onset of paralysis in the legs a few hours after taking the drug, no sensory involvement, by paralysis and wasting of the opponens pollucis and interossei, and no recovery after 6 to 8 months. CLINICAL CLASSIFICATION OF POLYNEURITIS Total Polyneuritis 70 1. Diphtheritic nouritis 42 Certified dip#, typical neuritis 21 Probable dip., typical neuritis 14 Skin dip., typical neuritis 5 Probable dip., incomplete neuritis 7 2. Sulfonamide neuritis 15 3. Post-infectious neuritis 6 (Wound sepsis) 4. Unclassified neuritis 9 No sore throat, atypical neuritis 5 Sore throat, incomplete neuritis 4 CONTROLS 112 Recovering diphtheria 17 More than 4 weeks after serum 8 Less than 4 weeks after serum 9 TOTAL 199 October 15, 1945 Clinical Classification and Results of Cultural and Serologic Studies of Polyneuritis Cases at Merano, Italy, July 1945 Clinical classifi- cation No. No. Culture + virulent C Antitoxin content of . serum Cult. neg. for vir. C diphtheria. Antitoxin content of serum *>y stud- cul- diphtheria XOl unit 1 >.01 unit <.01 unit l>.01 unit Dr. Gammon ied tured No. % No. i No. fo No. $ r No.'' Diph.Poly- neuritis 42 42 5 11.9 23 54.6 19 i 45.4 22 59.6 40.4 Sulfon- amide Poly neuritis 13 13 0 0.0 5 38.4 8 61.6 5 38.4 i [ 8 61.6 Post- infec- tious Poly- neuritis 6 6 2 2 4 2 2 Un- classified Poly- neuritis 9 9 3 3 6 3 3 Total Poly neuritis m 70 70 10 14.2 33 48.6 37 51.4 32 53.4 28 46.6 Control Group No Diph- theria No Poly- neuritis 112 107 18 16.9 25 22.4 87 77.6 20 22.4 69 77.6 Con- valescent Diphtheria 17 17 9 52.6 5 12 i 1 7 a) More than 4 weeks 8 after therapy 8 4 50.0 4 4 1 3 5, VACCINATION OF HUMAN BEINGS WITH DENGUE VIRUS MODIFIED BY PASSAGE IN MICE by Lt. Col* A. B# Sabin, M. C. and Capt. R. W. Schlesinger, M. C. It was reported at the last meeting of the Commission that the results of tests on 9 human volunteers inoculated with the 7th to the 10th mouse passage virus indicated that the virus had undergone sufficient attenuation to permit its consideration for use as a vaccine against dengue. During the past S months two vaccines, prepared respectively from 15th and 19th mouse passage virus, were tested both in AST? medical student volunteers and schizophrenic patients# The 15th passage vaccine, consisting of a centrifuged 1:10 mouse brain extract in 10 per cent human serum-saline solution, was tested in 10 students and 6 schizo- phrenic patients in doses of 0.5 cc. of the It 100, 1:1,000, and 1:10,000 dilutions. ( All of these developed a maculo-papular eruption of varying extent, while systemic symptoms were either absent, negligible or very mild; all were found to be immune upon exposure to Aedes aegypti mosquitoes of proved infectivity 21 to 38 days after vaccination# It was thus found that the extract from a single mouse brain and cord proservod in the frozen state had enough antigen in it to immunize at least 10,000 people. When some of the same 15th passage vaccine was lyophilizod together with yellow fever vaccine and tested in 10 students, it failed to produce immunity in at least half of those who wore exposed to the infected mosquitoes. An extensive test was carried out to determine the infectivity of Aedes aegypti mosquitoes feeding on people from one to 14 days after vaccination with the modified dengue virus. The engorged mosquitoes were allowed an extrinsic incuba- tion period of 29 to 33 days before different pools representing different days after vaccination were tested on 14 students. With 22 to 42 mosquitoes engorg- ing in the tests on each volunteer, there was no evidence of dengue infection in any of the 14 men# Since it desirable to get away from human serum as a constituent of the vaccine, ciystalline bovine albumin was selected as the protein to be used for both extraction and lyophilization in the tests with the 19th mouse passage vaccine. A relatively large amount of vaccine was prepared both as a model for production on a still larger scale and for the purpose of having enough available for a field trial if the preliminary human tests were satisfactory. The vaccine was lyophilized in two forms—as 10 per cent and as 1 per cent extract of mouse brain and cord in 10 per cent crystalline bovine albumin. Simultaneous titrations in mice of the frozen and lyophilized 10 per cent vaccines revealed that the lyophilized material had only l/lO as much virus as the frozen preparation. Human tests on 15 subjects (8 students and 7 schizophrenic patients) revealed that the 10 per cent lyophilized vaccine produced the same type of reactions as the 16th passage frozen vaccine except that in the 1*10,000 dilution, the lyophilized preparation produced rash in only 1 of 5 subjects and immunity to a large dose of unmodified dengue virus (perhaps as much as 1,000,000 human M. I.D.) in 2 of the 3 subjects. The 1 per lyophilized vaccine was apparently ineffective since neither rash nor immunity resulted from the 1*1,000 or 1*10,000 doses. It is believed that this 19th mouse passage lyophilized dengue virus is suitable for a trial of its protective capacity during an epidemic of the disease. Enough of this vaccine is now on hand to permit such a trial on a largo scale. 6. DENGUE VIRUS IN CHICK EMBRYOS by Capt. R. W. Schlesingor, M.C. and Lt. Col. A. B. Sabin, M.C. Wo have previously reported that various attempts to propagate unmodi- fied dengue virus in ombryonated eggs or tissue culture media yielded negative results. More recent tests in which the mouse-adapted Hawaiian strain was used as starting inoculum can be summarized as follows* 1. Attempts to propagate the mouse-adapted virus, using passages IV, V and XIII, in fluid or plasma clot cultures containing mouse embryo brain tissue wore unsuccessful in that no virus pathogenic for mico was demonstrable after one, two or throe passages. 2» When the 13th passage mouse-adapted virus was used for inoculation of 6~ or 10-dcy old chick embryos which wore subsequently incubated for 8 or 4 days respectively at 37°C, no mouse-pathogcnic virus was demonstrable after one to four serial passages* The results were the same when the Inocula were introduced into the yolk sac or embryo, allantoic or amniotic sacs* 3# Mien the 13th passage mouse-adapted virus was inoculated directly into the brain cf 10-day old embryos vdiich were subsequently incubated for V days o at 37 C, mouse-pathogenic virus was demonstrable in the chick embryo brain tissue in the first passage, but not in the second. Repetition of this pro- cedure with 16th passage mouse-adapted virus yielded negative results even in the first passage. 4. When the 16th or 18th passage mouse-adapted virus was used for inoculation (toward the embryo) of 5-day old chick embryos which were subse- quently incubated for 8 days at 37° C, mouse-pathogenic virus was demonstrable in whole embryo extract of the first passage in 3 separate series. In only one of these series, however, was virus demonstrable in the second passage, but even in this series it was no longer present in the 3rd passage when the incubation was at 37°C. It was found, however, that incubation at 36°C was more suitable for the serial propagation of the virus, so that it has been possible to demonstrate appreciable amounts of virus in the 3rd passage of at least 2 series. There is some indication that 4 or 5 days of incubation is not as good as 8 days, and that while the virus is present in the amniotic membrane as well as in the whole embryo, little or no virus has been found in the amniotic fluid* 5. The pathogenicity of this chick-embryo passaged virus for human beings is now under investigation. 6* The results of the tests on human beings which became available several days after tho meeting, indicated that the chick embryo material which was pathogenic for mice, produced the same clinical manifestations as the mouse passaged dengue virus. 7. DENGUE NEUTRALIZATION TESTS IN MICE* by Capt. R. W. Schlosinger, H.C. and Lt. Col* A* B* Sabin, M.C. The presence of type-specific neutralizing antibodies in dengue oon*- valescent sera demonstrable by tests on human beings was previously reported to the Commission. When the mouse-adapted virus had attained sufficient virulence to produce clinically apparent infection in almost all mice inoculated with a 10 per cent mouse brain suspension, investigations were undertaken to determine whether or not neutralization tests could bo carried out in mice. It has been found thus far, that when five to ton 50 per cent infective doses are used, the neutralizing effect is demonstrable by absence of clini- cal signs and survival of the mice, whereas with larger amounts of virus the only effect may be a prolongation in the incubation period* The marked depend- ence of the test on the quantity of virus used has caused considerable diffi- culty especially since the virus has been increasing in titre recently* The results obtained thus far indicate that, as in the human tests, the neutraliz- ing effect of dengue convalescent scrum in mice is type-specific* In its present stage of development the mouse test seems, however, to bo much less sensitive than the human test. Further work is necessary to determine the best way to carry out the tost, especially with more virulent mouse virus, before any decision con be reached regarding its possible use in diagnosis or epidemiological investigations. 8. REPORT OF COMMITTEE ON NEUTRALIZATION TESTS by Dr. P. K. Olitsty* Dr* Olitsky stated that his Committee had considered the inadequacy of the directions issued by the previous committee on neutralization tests and had discussed at some length methods of improving the situation* He believed that owing to the complexity of the problem and the fact that the testa vary greatly with different viruses it now seemed unwise for the Commission to champion any special technique unless there ms a demand on the part of certain Army laboratories for such directions. Col. Smadel inquired as to whether the Commission might not advise their laboratory on certain features which have been found to influence the tests, even though no techniques were recommended specifically. It was decided to inquire from the Surgeon General*s Office for advice as to whether a demand for directions in the performance of the neutraliza- tion test now exists. ♦9. THE CURRENT STATE OF PRODUCTION OF EQUINE ENCEPHALOMYELITIS VIRUS VACCINES by Dr. P.K. Olitsky. The Lederle Laboratories are now processing 30 liters of Western and 30 liters of Eastern virus vaccines for human use. However, more than 100 liters of veterinary vaccines are available. It is the opinion of Dr. Cox of the Lederle Laboratories that, in the face of sudden need for mass immun- ization, this vaccine can be diluted 1*1 and used for man. The veterinary vaccine differs from the human in that it contains 40 per cent tissue instead of 20 per cent; has 0.4 per cent formalin instead of 0.2 per cent; is not centrifuged, and has not the phenyl mercuric nitrate (1:12,500) used in the human product. But the same "identity"# potency and sterility tests are applied to both types. A current project is the determination of a means for increasing the stability of the vaccine since its immunogenic potency begins to fall after 8 to 10 months* storage. ♦10. RUSSIAN SPRING-SUMMER ENCEPHALITIS. FINAL REPORT ON VACCINATION STUDIES by Drs. J. Casals and P.K. Olitsky. Continuing studies on resistance to infection and antibody response following vaccination with Russian Spring-Summer (Tick-Borne) encephalitis virus [see report of Commission Meeting April 3, 19453 show 1) mice about 1 l/2 years old exhibited staunch immunity to peripheral (but not to intra- cerebral) inoculation of virus (to >8 million lethal doses) about 15 months after vaccination; 2) neutralizing antibody was then still high (serum neutralized more than 30,000 m.l#d#), as determined by intraperitoneal test but practically negative by the intracerebral route (neutralization of 8 m#l#d#)j 3) complement-fixing antibody was not detectable# A correlation therefore exists in mice between neutralizing antibody as determined by the intraperitoneal test and immunity to peripheral inocu- lation of the virus. ♦ 11# EFFECT OF NORMAL SERUM ON TITER OF VIRUS by Drs. J. Casals and P.K. Olitsky. Blood sera from several animal species, including the mouse, hamster, guinea-pig, monkey and rabbit, have the property, in the fresh state, of reducing the titer of certain neurotropic viruses, when the virus and serum are brought together under favorable conditions before inoculation# The nature of this inhibitory factor is being investigated. Thus far it has been found that the inhibition is a function of temperature and length of incubation# It does not depend on the route of inoculation, and heat at 56 to 60°C may or may not destroy part of the inhibitory effect# ♦12#DIAGNOSIS OF ENCEPHALITIS VIRUSES BY ANIMAL INOCULATION AND COMPLEMENT- FIXATION TESTS by Dr. J. Casals. A great deal of the work of this laboratory consists of making such tests at the request of the Commission, the Army and Navy, and others, and preparing and distributing standard sera and antigens for the tests# With respect to complement-fixation tests, a simple method by means of heat (60 to 62°C for l/2 hour) for inactivation of certain antigens for safe handling in laboratories has been devised. The Japanese B, West Nile, Russian spring-summer, St. Louis, and Western equine encephalitis and louping-ill antigens can be so treated successfully, but not lymphocytic choriomeningitis or Venezuelan equine antigens. In the case of the Eastern equino encephalitis antigen, the value of this method is dubious* During the last six months, about 100 sera received from Army, Navy, and civilian sources, both here and abroad, have been sent to us for com- plement-fixation tests. All were negative with different encephalitis virus antigens except one, a Canadian serum, which was positive for lymphocytic choriomeningitis. Twenty spinal fluids and two brain specimens were re- ceived, Animal inoculation tests were negative in all instances. About 1,000 cc, of lyophilized, inactivated antigens have been dis- tributed, along with samples of standard antisera against different viruses, exclusively to the Army and Navy. 13, EXPERIMENTS IN INFECTIOUS HEPATITIS by Maj. W.P, Havens, Jr., M.C. Recent experiments with infectious hepatitis carried out at the Com- mission* s laboratory at the Yale University Medical School have dealt with the following points: 1: Determination of the period of infoctivity of patients with the disease. 2: Determination of the infoctivity of urine and na so-pharyngeal washings obtained in the acute phase of infectious hepatitis. 3: Determination of whether patients with infectious hepatitis induced by parenteral inoculation eliminate virus in the stool. 4s Hematologic changes appearing during the incubation period and course of infectious hepatitis in human volunteers. 5: Roentgenologic and gastroscopic findings in volunteers with infectious hepatitis, 6; Attempts to transmit infectious hepatitis to chimpanzees. The strain of infectious hepatitis virus used in all the experiments was originally derived from the stool of a U. S. soldier who contracted epidemic hepatitis in Sicily In September 1943. This virus has been through four passages in human subjects. It is filtrable, heat resistant, and is regularly present in the stool and serum of patients in the acute, pre-icteric phase of the disease. It has produced infectious hepatitis in 70% of human volunteers following ingestion or parenteral inoculation with incubation periods ranging from 15 to 34 days. 1# Infactivity Test?.- (a) Although acute-phase serum and stool of volunteers with experimentally induced infectious hepatitis are known to be infectious, the period of infectivity has not been previously defined. Serum and stools were obtained therefore during the incubation period, the pre-icteric phase of the disease, and in convalescence, i.e., 28 days after onset of disease, in one volunteer who had contracted infectious hepatitis 24 days after ingesting infectious serum. Serum from this patient was inocu- lated parenterally and his stool was fed to human volunteers. The infectivity of the acute-phase serum and stool was confirmed by positive tests in 5 of 9 human volunteers who received those materials. Neither serum obtained mid- way through the incubation period nor stools in the convalescent phase of this one volunteer were infectious. Dosages varying from 0.01 to 0.5 cc. of infectious serum (acute phase) caused no significant variation of incubation period. (B) This experiment was repeated using a pool of material consisting of acute-phase serum,stools, urine, and naso-pharyngeal washings, and con- valescent stools, from five patients with experimentally induced infectious hepatitis which had resulted from feeding infectious materials. The infec- tivity of the acute-phase serum and stools was again evident, with 5 of 6 subjects who received this material, contracting the disease. Both urine and naso-pharyngeal washings failed to produce the disease in 6 volunteers. Convalescent phase stools were again negative in four subjects. (C) Although it is known that patients with both naturally occurring infectious hepatitis and infectious hepatitis experimentally induced by feeding infectious materials, eliminate virus in the stool, it had not been determined whether patients with experimental infectious hepatitis induced by parenteral inoculation also eliminate virus in the stool. This is of collateral interest in attempting to relate the naturally occurring disease to homologous serum jaundice for in the latter condition, which has been produced experimentally only by parenteral inoculation (with the exception of the single transmission to a human subject effected by MacCallum and Bauer by feeding the icterogenio agent), virus has never been recovered from the stool* Acute-phase serum and stool were pooled from 8 volunteers whose disease had been induced by parenteral inoculation of our strain of infec- tious hepatitis virus* All of these men had had short incubation periods ranging from 20 to 30 days. Virus was eliminated in the stools of such patients during the acute phase of the disease, producing infectious hepatitis in 3 out of 3 volunteers after ingestion* It was also present in the acute- phase scrum, producing tho disease in 2 out of 3 volunteers* 2* Hematology*- It has been pointed out for several years by American and British workers that atypical mononuclear cells appear in the peripheral blood during tho course of infectious hepatitis* Systematic leukocyte counts on our experimental infectious hepatitis subjects have revealed a regular pattern of change, characterised by the early appearance of leukopenia ’with both absolute and relative lymphopenia and relative polymorphonuclear leukocytosis. Subsequently the neutrophils are depressed to such a low level that a relative lymphocytosis exists. Although minor changes may occur 2-3 days before the onset of fever, the striking changes are primarily associated with fever. Those patients whose onset is acute with fever, manifest the hematologic changes within the first 24 to 36 hours of disease while those whose onset is gradual with vague generalized or abdominal complaints, have the characteristic blood changes several days later coincident with tho beginning of fever. Tho degree of change in the blood studies apparently parallels the severity of the febrile response* In all but one patient, leukopenia, i*e., a reduction to below 6000 lcukooytes/cu,mm*, was found at some time during the pro-icteric acute phase of the disease* Of interest has been the appearance of the lymphocytes during the acute phase* As early as the first or second day when lymphopenia is pro- nounced an occasional atypical, lymphocyte is seen. Such cells are character- ized by their large size and characteristic changes in the nucleus and cytoplasm. They resemble cells seen in infectious mononucleosis. These cells increase in percentage and absolute numbers until the fifth to seventh day of fever when lymphocytosis is present. At this time they may number as many as 18 to 20 per cent of the total lymphocyte count. By the tenth day -when the lymphocyte count has assumed normal proportions of the total count, these atypical lymphocytes have largely disappeared. Their appearance is correlated in many patients with the appearance of posterior cervical adenitis. 3. Gastroscopy.- (with Dr. S. Kushlan and Dr. R, Green) Gastro- scopic examination of patients with infectious hepatitis by Knight and Cogs- well showed changes from normal in the appearance of the gastric mucosa in 7 out of 9 patients during early pre-icteric and icteric phases. The find- ings were described as those generally interpreted as superficial gastritis involving primarily the antral portions of the stomach and in some instances the fundus. In five of the patients small aphthous ulcers from two to five j? n millimeters in diameter were reported in the antrum and o/ the angularis. In repeating this worl: on our experimentally induced cases we have had an opportunity to study the appearance of the gastric mucosa before, during, and after the course of the disease. Six men had gastroscopic exami- nations before inoculation,during illness, and after recovery (2 months after onset) from infectious hepatitis. During the disease gastroscopy was per- formed from the third to the seventeenth day of illness. Of the six patients who were gastroscoped before, during, and after experimentally induced infectious hepatitis, two showed unequivocal changes. These two (SIT and TR) revealed perfectly normal mucosa in the antrum and body of the stomach, both before infection and after complete subsidence of the infection. The changes during the acute phase of disease were the same in both patients, differing only in degree, and consisted of a marked crim- son red appearance with edema, both more acute in SN who was examined earlier in the illness (pre-icteric) than in TR (7th day of jaundice)# In another man (WIT), the changes seen were suggestive* Prior to infection, the gastric mucosa appeared normal* During his illness, the mucosa was fiery red and edematous as in the last two. However, on the final post- illness control examination, similar changes were noted but the redness was less fiery, bordering on an orange-red; edema was less marked. This would seem to represent a subsiding superficial gastritis. In the other 3 oases, the changes were entirely equivocal and from a gastroscopic viewpoint were inconclusive* 4* Roentgenograms*- X-rays of the gastro-intestinal tract after an opaque meal, were made in 12 subjects before inoculation and from 10 to 17 days after onset of infectious hepatitis# It was concluded that no definite constant pathology was determined by radiological examination of those 12 subjects examined before inoculation and during the acute phase of infectious i hepatitis# 5# Attempts to infect 6 chimpanzees (2 adults, 4 young) with proven infectious hepatitis virus were unsuccessful* 14* SERUM JAUNDICE by Dr. C. A. Jane way. 1. Frequency in a Civilian Hospital.- Observations on the frequency of jaundice following the transfusion of blood and plasma procured and issued through the blood bank of a Boston hospital indicate that this disease occurs more frequently in this country than is usually realized. The usual plasma pool in this blood bank is made up from an average of 16 bloodings* One plasma pool (#219) is of particular interest. Of the seven recipients who survived long enough to develop jaundice, four (all adults) developed hepatitis approximately 90 after transfusion and one died. Th~ fifth and sixth recipients, aged 7 and 19 months escaped. All the deno: s to the pool havo been traced and questioned by interview if possible or else by nail; except two, who are in the Navy and are now being traced. Of the 13 donors questioned, two have interesting histories. One is known to have congeni- tal hemolytic jaundice and had a splenectomy. Her child was having a splenectomy for that condition at the time of her donation, Tho other has a much more suspicious history. Four years before his donation, he and his wife had "intestinal grippe with a touch of Jaundice." Although the donor has been well since, his wife has had recurrences of intestinal grippe with slight jaundice every winter since then, 2, Serum Jaundice in Relation to Plasma Fractionation,- Two features of the process of plasma fractionation would appear to favor tho dissemina- tion of hepatitis virus in its products; First, the largo size of the plasma pools (250-10,000 bleedings), and second, the gentleness of the fractionation procedure which is carried out using alcohol as a precipitant at temperatures below freezing point of water. Studies by Drs. W, C, Boyd and J. F. Enders showed that two viruses (Theilerfs mouse encephalomyelitis virus and tobacco mosaic virus) added to the starting plasma could be detected in all tho major fractions. For this reason an attempt has been made to follow as many patients as possible who have received albumin and gamma-globulin, Tho re- sults indicate that up to the present these two products have probably not transmitted hepatitis to any recipients, 3, Thermal Death Time of Agent of Serum Jaundice. Because tho possi- bility of transmission of hepatitis by human serum albumin had not been eliminated, it was decided to determine whether the virus could be killed by heat In the presence of albumin. An experiment has been carried out by Dr, George Scatchard, Dr, L, E. Strong, ond myself, working in collaboration with Dr, Joseph Stokes and Capt, Sidney Gellis of the Commission on Measles and Mumps. On tho basis of Dr. J, Murray Luck’s discovery that nonpolar anions would increase tho thermal stability of albumin, it has been possible to develop a low salt concentrated albumin solution, stabilized with either .04 M, aootyl tryptophanate or a mixture of ,02 M acetyl tryptophanate and ,02 M caprylato at a pH of G,8“0*2, The first solution can be heated to a tem- perature of 60°C for approximately 18 to 20 hours, the latter at 64°C for approximately the same period, before visible turbidity develops, A sample of Fort Bragg plasma (derived from cases of yellow fever vaccine jaundice) was added in a proportion of 1 volume of plasma to 4 volumes of concentrated albumin solution. Three aliquots of this mixture were treated as follows; A - (Albumin with ,04 M acetyl tryptophanate) held in the cold room, for control, B - (Albumin with ,04 M acetyl tryptophanate) heated 10 hours at 60°C in a mi ter bath, C - (Albumin with ,02 M acetyl tryptophanate and ,02 M caprylato) heated 10 hours at 64°C in a water bath. At the end of the heating period, the three mixtures were frozen at 70°C, packed in dry ice and shipped to Capt, Gellis, who administered them by intramuscular injection to a group of volunteers the following day, Bach man received an inoculum of 10 cc. containing 2 cc, of plasma, except the controls who received approximately half this dose. Results: Although the experiment is not completed (15 weeks have elapsed), so far five out of ten controls have developed hepatitis, while, of the five men in each group receiving mixtures B and C, none have developed any evidence of hepatitis, 15. INFECTIOUS HEPATITIS. LESIONS IN LIVERS OF NORMAL MICE by Drs, p. K. Olitsky and J, Casals, In normal. Rockefeller Institute or Swiss strains of albino mice, certain spontaneous lesions were observed -which should bo considered in con- nection with attempts to transmit liver affections to this animal. One type consists of infiltration about the bile ducts, also about blood vessels, of polymorphonuclear (predominantly) along with monocytic, lymphocytic and plasma cells* Associated is a focal necrosis of individual hepatic cells* Another type is characterized by the presence of intranuclear inclusion bodies. The relation of one type of lesion to the other is as yet undotor- Tinned; the inclusion bodies are found only in older nice and the infiltrative lesion is noted especially with increasing age. There is some resemblance between the picture of the inclusions to those reported by Findlay only in p, particular (Clacton) strain of mice; their identity is a matter for further study# Liver Function Test in Mice.- Recently Maclagen described a new test for liver function in man, the thymol turbidity test. In connection with the spontaneous lesions found in mice, as described above, we undertook an investigation of the value of this test applied to normal mice and to mice having pathological conditions in the liver spontaneously developed or arti- ficially induced# Thus far a normal base line for this animal has been established. In the discussion Capt. Schlesinger reported spontaneous jaundice occurring in 1 mouse whose serum bilirubin was 20 mg. per cent# Sections showed necrosis of liver cells# Passage to mice and other animals ms unsuccessful. 16. POLIOMYELITIS IN 194-5# THE OUTBREAK AT FORT McCLELLAN. ALABAMA by Dr. R* Ward. An unusual, sharp and moderately serious outbreak of 17 cases of poliomyelitis occurred at Fort McClellan, Alabama, during March and April 1945# Among these 17 cases there were 10 with bulbar symptoms; 3 of these were fatal# An investigation was carried out to determine if possible, what factors were involved in the dissemination of virus through this camp. The oases were grouped rather closely in point of time of onset, but scat- tered throughout the comp from a geographical standpoint. Their chronological grouping suggested that r. large amount of virus was rathe" suddenly introduced 29 by unknown channels into the camp population. The spotty distribution of cases throughout the camp is compatible with the theory that there may have been a very wide dissemination of virus to which many could have been ex- posed but only an occasional susceptible acquired the disease among this fairly resistant population of adults. The onset of the epidemic was heralded by a single diagnosed case which preceded the others by 3 or more weeks. He and/or other undiagnosed cases or carriers, might have been entirely re- sponsible for spreading the virus throughout the camp# We believe this to be unlikely* It is pertinent to call attention to the fact that a soldier at the nearby but much smaller Camp Sibert (about 40 miles distant) also contracted tr poliomyelitis during the period of the epidemic at fort McClellan. The Camp Sibert patient had not visited Fort McClellan. Mention of this fact is made to indicate that intrinsic factors of spread at Fort McClellan may not have been entirely responsible for the prevalence of poliomyelitis in this fairly small area of two counties. Three civilian oases were also recorded in nearby counties in the month preceding the camp epidemic. The investigation has been conducted with the idea that the manner of spread of poliomyelitis in anv epidemic is unknown. A number of different modes of spread of poliomyelitis virus deserve consideration in the Fort McClellan outbreak and at Camp Sibert. Infection might have been entirely the result of exposure to infective human carriers, or to contaminated food, or milk or ice cream, as well as to some other extra-human source of infection. Contamination of water supply seems unlikely. Tests carried out on water, milk, ice cream, sludge and flies were negative for poliomyelitis virus. Although no definite answer can be made as to the manner of spread in this outbreak, it is pointed out that epidemics of poliomyelitis in Any camps, occurring as they do in a population which is more controlled than in civilian populations of the same size, offer certain opportunities which should be of considerable value in tracing the spread of this disease if enough epidemics of this type are adequately studied. 17. CIVILIAN EPIDEMICS OF POLIOMYELITIS IN THE EAST - 1945 by Dr. J.R, Paul. Individual epidemics were not enumerated but a few items seen during the past summer were mentioned. (1) The occurrence in July 1945 during the epidemic at Paterson, N.J., of a severe flood within the epidemic urban area during the period in •■which the epidemic was getting under way. The flood inundated about l/lO of the city and caused sewage to be floated into many city streets at a time when there must have been many intestinal carriers of virus in the city. The subsequent course of the epidemic did not seem to be influenced in any par- ticular direction by this flood, (2) Attention was called to the high prevalence of bulbar cases and the high mortality (similar to the epidemic at Fort McClellan) which was noted in the epidemic at Rockford, 111., in July and August 1945. (3) Mention was made of the death of a technician working with recent- ly isolated strains of virus in the Yale Poliomyelitis Study Unit* The fact that two such laboratory accidents have now occurred, would indicate that exposure to ’’human” poliomyelitis virus in the laboratory is probably more dangerous than has been originally suspected. (4) Mention was made of a family of 8 children seen in August 1945 at Rockford, 111.,in which 8 cases of illness occurred including: 1 death, 3 paralytic cases and 4 abortive or questionable cases, Brents which trans- pired in this family offered an unusual opportunity for contrasting the early with late symptoms of the clinical disease. 18. ENCEPHALITIS AND POLIOMYELITIS IN THE WEST by Dr. W. McD. Hammon. A. Kern County, California, 1944, Final Report.- The list of apparent neurotropic virus infections seen at the Kern County General Hospital was finally reduced to 57. These con bo divided now into 6 groups: Group I, in 17 cases because of unsatisfactory specimens no etiologic diagnosis could be made. Group II, 4 cases of St, Louis (St. L.) encephalitis were diagnosed 31 by showing an increase in the antibody titer* Group III, 2 cases of Western equine (W.E.) type encephalitis were diagnosed by demonstrating an increase in titer* Group IV, 4 patients had flaccid paralysis; their fecej wore tested in monkeys with negative results. Group V, the 54 remaining patients, although many of them had antibodies to either the St* L* or the W.E. virus, were shown to have no increase in antibody titer to either virus during the period of the illness* Feces from 16 out of the 34 patients were tested in nwnkeys for poliomyelitis virus, but none was isolated. In view of the failure to detect poliomyelitis virus by methods which were successful with feces from the same region the previous year and from other regions during the same year, we do not feel that the 58 cases in the last 2 groups necessarily represent poliomyelitis. B* The California Virus*- Immunological tests show quite conclusively that the California virus is not similar to or closely related to Theiler's (TO) or Theiler’s GD-VII viruses, nor to the 5 Columbian viruses. Anopheles A, Anopheles B, or T