原著：「Propagation in  Tissue  Cultures  of  Cytopathogenic  Agents  from  Patients with  Measles」（PDF）

邦訳：「麻疹患者からの細胞変性病原体の組織培養による増殖」（PDF）

著　者：J. Enders, T. Peebles

掲載年：1954年6月1日

出版元：Medicine, Biology

掲載誌：Proceedings of the Society for Experimental Biology and Medicine

JOHN F. ENDERS  AND  THOMAS C. PEEBLE
(With the assistance of  Yinette Chang and Ann Holloway.)

From  the  Researah  Division  of  Infectious  Diseases,  Children's  Medical  Center, Boston,  Mass.  and Departments  of  Bacteriology  and  Immunology  and  of  Pediatrics,  Harvard  Medical  School

Numerous attempts have been made in the past  to  propagate  the  agent  of  measles  in lower animals, in chick embryos and in  tissue cultures(1-3). The  results  of  different  investigators were often  at variance or  directly contradictory. It has  been  made  reasonably clear,  however,  that  monkeys,  especially M. mulatta, are moderately susceptible to experimental  inoculation (3). Furthermore  the  researches of  Rake, Shaffer and their collaborators  have  provided  evidence  suggesting  that the  agent  which  passed  through  bacteria-retaining filters could be maintained indefinitely in  serial  passages  in  the  developing  chick embryo(4, 5). These  workers (5)   also  confirmed  the  earlier  observations  of  Plotz(6)  who apparently had succeeded in growing the agent in a modified suspended cell  culture of chick embryonic tissues. Egg passage in  the hands of Shaffer and his coworkers(7,8) regularly  appeared  to  alter  the  pathogenicity  of the agent for man as indicated by the development of  a mild and much modified disease following  the  inoculation  of  egg  adapted  materials  into  susceptible children. In  certain cases this  modified disease seemed to  be  followed  by  resistance  to  measles  as  indicated by  the  results  of  subsequent natural  or  artificial  exposure  to  the  virulent  form  of  the agent(9). Since  1943  when  the  last  of  the communications by Rake and his collaborators appeared,  no  important  progress  has  been made in  the study of  the etiology of  measles. This  fact  may  in  large part  be  attributed  to the  lack  of  a  convenient  laboratory  method for the  demonstration of  the presence of  the agent which  induced no  recognizable changes in  eggs  or  cultures  of  chick  tissues. Moreover,  repeated  attempts  by  Shaffer(10)  to demonstrate a serologic reaction, such as complement  fixation,  using  materials  from  the infected  chick  embryo  failed. Accordingly, the  only  available technics have  consisted in the  inoculation of  man  or  the  monkey. The former is obviously impractical as routine and the  latter  tedious,  expensive  and  frequently inconclusive because of  variation in individual susceptibility.

With these considerations in mind we have recently  attempted  to  cultivate  the  agent  of measles  in  cultures  of  human  and  monkey cells  employing  procedures  applied  successfully  to  the  propagation  of  the  poliomyelitis viruses(11-13). In blood and throat washings of  typical  cases of  measles agents have been demonstrated that can be maintained in serial passage  in  tissue  cultures  and  which  induce distinctive  cytopathic  changes  in  renal  epithelial  cells. A  certain  amount  of  evidence has  been  accumulated  indicating  that  antibodies specific for these agents develop during
the  course  of  the  disease. It is  our  purpose to  describe  here  these  observations  in  a  preliminary manner. Additional evidence for the relationship  of  these  agents  to  measles  will be  sought  in  future investigations.

TABLE  I.

Cytopathogenic  Agents  Isolated  in Tissue  Cultures  from  Throat  Washings  and  Blood of  5  Measles  Cases.

Collection of  specimens.

Throat  washings,  venous  blood  and feces were obtained from 7 patients as early as possible  after  a  clinical  diagnosis  of  measles was  established. In  5  instances  the  time at  which  specimens  were  collected  in  relation  to  the  onset  of  exanthem  is  given in  the  case  histories  described  below  or in  Table  I. When  capable,  patients  were asked  to  gargle  with  10-15  ml  of  sterile neutralized  fat-free  milk.
Certain  specimens  from  the  throats  of  younger  children were obtained by cotton swab previously moistened in milk. After swabbing the throat the swab was immersed in 2 ml of  milk. Penicillin, 100 u/ml,  and streptomycin, 50 mg/ml, were added to all throat specimens which were then  centrifuged  at  5450  rpm  for  about  one hour. Supernatant fluid  and  sediment  resuspended in a small volume of  milk were used as separate  inocula  in  different  experiments  in amounts varying from 0.5 ml to 3.0 ml. About 10 ml  of  blood  immediately  after  withdrawal were placed in tubes containing  2  ml of  0.05% solution of  heparin. As inocula for tissue cultures amounts varying from  0.5 ml to 2.0  ml of  the  whole  blood  were  employed. After addition of  antibiotics as described above 10% fecal  suspensions  were  prepared  by  grinding the material in bovine  amniotic fluid medium. The  suspensions  were  then  centrifuged  at 5450 rpm  for about  one  hour  and  the  supernatant  fluids  used  as  inocula,  in  amounts varying  from  0.1  ml  to  3  ml. All  specimens were  refrigerated  in  water  and  ice  or  maintained  in the cold at about 5°C from the time of  collection  until  they  were  added  to  the cultures. The  maximum  time  that  lapsed between  collection  of  specimens  and  inoculation  was 3 1/2  hours. 

In the initial isolation  attempts  roller  tube  cultures (11, 12)   of human kidney, human embryonic lung, human embryonic  intestine,  human uterus and rhesus monkey  testis  were  employed. Subsequent passages  of  the agents isolated  were  later  attempted  in  human  kidney,  human  embryonic skin  and  muscle,  human  foreskin,  human uterus,  rhesus  monkey  kidney and embryonic chick tissue. Stationary cultures prepared according  to  the  technic  of  Youngner(13) with trypsinized  human and rhesus monkey kidney were later employed for isolation of  agents and their passage. The culture medium  consisted of  bovine  amniotic fluid  (90%),  beef  embryo extract  (5%), horse serum  (5 %) ,   antibiotics, and  phenol  red  as  an  indicator  of  cell  metabolism (12). Soybean  trypsin inhibitor was added to this medium  unless  it was  used  for the cultivation of  human and monkey kidney (11). Fluids were  usually  changed  at intervals  of  4-5  days. For  histological  examination  the  cell  growth  after  fixation  in  10% formalin  was  embedded  in  collodion,  dehydrated  and  stained  with  hematoxylin  and eosin. 

Serial passage  of  the  various  strains  (Table I) was accomplished  as  routine  by  removal  of  the culture medium between the 4th and the 16th day after  inoculation  and  immediate  transfer of 0.1 ml to each of  a number of  fresh cultures. Successful  passage  of  the  agent  with  fluids that had  been  previously  centrifuged  at 2500 rpm to remove cellular elements has also been repeatedly demonstrated. Larger inocula  (up to  1.0 ml)  were  often used  during  the  initial experiments  before the resistance of  the agent to  storage  at various  temperatures  had  been determined.

Virus neutralization  and complement fixation tests.

The procedures employed are described  subsequently  in  the  text.

Description  of  cases  from  which  materials  were obtained.

During  an outbreak  of  measles at a  private  boarding  school for  boys  in  Southboro,  Mass.,  isolations  were  attempted  from throat washings  and  stools  of  4 patients  and blood of 3 of  the same patients. The latter are designated as Cases 1, 2  and 3. Throat washings  of  2  siblings  in  a  small  epidemic  of measles in Wellesley, Mass.,  (Cases 4 and  5) and blood from 2  cases of measles admitted to the  Boston  City Hospital  during  an epidemic in Boston  (Cases 6 and  7)   are under  investigation  at the present  time. Details  of  these typical  cases  are  omitted  for  the  sake  of brevity.

D.R., age 11, was in contact with .a "probable"  case  of  measles  10  days  before symptoms  commenced  on  1/20/54. The latter  consisted  of  signs  of  an upper  respiratory  infection  including  sore throat  and  fever to  101°F. He soon  developed  conjunctivitis and  a  bad  cough. These  symptoms  became aggravated  and on  1/24 in the morning  there was a suggestion of  a rash on his face. Temperature at 4:30 p.m. was  105.5°F. Koplik's spots were noted on the buccal mucosa by the school physician  the next morning. On  1/25 at  1 p.m.  he was seen by TCP with  findings of  temperature  98°  p.o.,  mild  to  moderate conjunctivitis,  moderate  generalized  adenopathy  and  characteristic  blotchy  maculopapular  rash  in  full  bloom,  extending  to  involve even the palms and  soles. No Koplik's spots  were  seen  at this  time. Specimens of throat  washings,  blood  and  stool  were  collected  at 1:30 p.m. on  1/25.

H.J., age  13,  with  no  known  contact  other  than Case 1, developed signs of  an upper respiratory infection  on 1/26/54. He  complained  of sore  throat and  cough  the  following day and of  photophobia  on  1/28.
At  this  time  the infirmary  nurse  noticed  a  questionable  trace of  a  rash  on  his  forehead. He was  seen  on 1/29  by  TCP  with  findings  of  temperature 101° p.o., and maculopapular rash developing over  the  face, ildly  over  the  chest  and  abdomen,  minimally  on  the  upper  extremities and  none  on  the  lower  extremities. He  had moderate  conjunctivitis,  cough  and' Koplik's spots  on  the  buccal  mucosa. Specimens  of throat  washings,  blood  and  stool  were  obtained at 11 a.m. on 1/29.

D.E., age 13,  a  close  contact  of  both  Cases  1  and  2 during  their  prodromal  stages,  experienced  a gastrointestinal  upset  on  2/8/54  with  cramps and nausea. Temperature was 99.6°F. Faint rash  was  first  noted  on  his  face  in  the  late evening of  2/9,  and  he was  seen by  TCP at 12 noon on 2/10  with findings of  typical  rash over  the  face  with  minimal  extension  to  the chest,  abdomen  and  back. Koplik's  spots were  present  on  the  buccal  mucosa. Cough and  conjunctivitis  were  mild. His  temperature was 102° p.o.
Specimens of  throat washings,  blood  and  stool  were  obtained  at  this time. His  temperature  was  103.6°F  with rash  in  full  bloom  in  the  afternoon. On  the following morning his temperature was  104°F.

FIG. 1.

Outgrowth of  normal huimn kidney cells in a n  uninoculated roller tube culture (X 130).  Control for cultures shown in  Fig. 2  and  3.

FIG. 2.

Area  of  syncytial giant cells with  small cytoplasmic  vacuoles. Note many  nucleoli  and faint  nuclear  outlines. 9th  day  after  inoculation;  7th  passage,  agent  from  Case  3  blood (X 130) . 

Cytopathic changes induced by agents isolated from cases of measles.

The first of  8 agents obtained from blood or throat washings of measles cases and exhibiting comparable  properties  was  isolated  in  cultures  of human kidney tissue following addition of  the blood of Case 3. In each of  the 3 cultures that were  inoculated  cytopathic  changes  were  observed  on  the  7th  day. Since  these  changes presented a characteristic appearance not heretofore  associated  definitely with  a  virus  they have provided  the  means  for  the  further investigation  of  this  agent  as  well  as  others that have been recently isolated. Accordingly, here  at  the  beginning  these  changes  will  be described  in  detail. Observation  of  fresh preparations  under  low  magnification  (80X) revealed  within  the  sheet-like  outgrowth  of renal  epithelial  cells discrete  areas of  varying size  and  shape  in  which  the  cell  boundaries were obliterated  and  the  nuclei  often  difficult to  visualize. Within  these  areas,  which  may be  described  as  non-refractile  "glassy" plaques,  large  and  small  vacuoles were  often numerous  lending  them  a  foamy or  lace-like quality. The number and size of  the vacuoles increased  as  incubation  was  continued. On careful examination of  these areas many small, slightly  refractile  bodies  were  seen  that  resembled nucleoli within  nuclei  whose  outlines could  often  be  distinguished  only  with  difficulty. The  total  effect  thus  suggested  the presence of  large vacuolated giant cells. After further cultivation the extent of  the areas initially  present  was  slowly  extended  or  was enlarged  by  coalescence  with  neighboring plaques  while  others developed  elsewhere. In addition  to  the  formation  of  vacuoles  degenerative  changes gradually  appeared  within the  affected  areas  suggesting  coagulation necrosis. At  the  end  of  three  weeks most  of the  epithelial  cells  appeared  to  be  involved, yet here and there small aggregates of  normal cells remained. These seemed, however, to be composed  mainly  of  spindle-shaped  cells. Reference  to  Fig. 1, 2  and  3  will  aid  in  the visualization  of  these  changes  as  they  are manifest  in  the  natural  state. In contrast  to the  appearance  of  the  normal  cell  outgrowth shown in  Fig. 1 the smooth  confluent area  of affected  cells  stands  out  clearly  in  Fig. 2. While  a  slight  degree  of  vacuolization  is evident  in  this  figure,  it  is  extensive  in  Fig. 3 especially  along  the  margin  of  cell  growth where it is first apt to become apparent.

The interpretation  that has  just  been  presented of  the changes observed in fresh preparations was supported by examination of  fixed and  stained  materials. Under  these  conditions the glassy areas were clearly  revealed as collections of  nuclei surrounded by a common cytoplasmic  matrix. As  many  as 40  to  100 nuclei were  counted  in  such  syncytial  formations. Often  the  limits  of  the  encompassing cytoplasm  were  sharply  defined  thus  contributing  to  the  impression  that  evelopment of  true  giant  cells  has  occurred  in  vitro. Whether  or  not  this  is actually  the  case,  the phenomenon is of  much interest in view of the constant  presence  of  giant  cells  in  lymphoid tissues  during  the  early  stages  of  measles  in man(14,15) .

Examination  of  stained  materials  also  revealed significant changes within  the nuclei of the  giant  cells that  were  not  visible  in  fresh preparations. These consisted in a redistribution  of  the  chromatin  which  ultimately  assumed  a  marginal  position  where  it  formed a dense ring or crescent that stairied intensely with  the  basic  dye. Concomitantly  the  central portion of  the nucleus came to be occupied by  an  apparently  homogeneous  substance, acidophilic  in  character,  that  approximated closely to the  chromatin  ring. Since in  these and  other  preparations  that  have  been  examined  subsequently no  clear  unstained  zone has been observed between  the chromatin and this  acidophilic  mass,  it  cannot  be  asserted that  the latter  represents  an  intranuclear  inclusion  body  of  the  type  characteristically associated  with  viral  infections. Nevertheless,  as  far  as  can  now  be  determined,  its presence  along  with  the  margination  of  the chromatin  affords  a useful  criterion  of  infection  for  the  agents  under  study. It  should be  emphasized,  however,  that  the  changes  as just depicted are encountered in cultures  that have  been  incubated  for  relatively  prolonged periods  (e.g. 14-21 days). When  the  interval between  inoculation  of  the  agent  and  examination  of  the  stained  cells  (e.g. 4  days)  is shorter,  margination  of  the  chromatin  may be  incomplete  or  inapparent  and  the  acidophilic  substance  may  only  be  seen  in  small rounded  masses  distributed  here  and  there amid  nuclear  materials  that approximate  the normal  arrangement.
Fig. 4, 5,  6  and 7  illustrate well-developed nuclear  changes and  also the general  similarity of  the  affected areas to the  giant  cells  encountered  in  lesions  associated with measles. Of  particular interest  in this latter connection are the basophilic “pseudoprotozoal”  bodies  that  Bonenfant (16)  has recently described in the mucosa and lymphoid follicles in cases of  this disease. These bodies were  usually  surrounded  by  an  acidophilic homogeneous  substance. As  described  and pictured  these bodies with  their  matrix  strikingly resemble the giant cells in tissue cultures that exhibit  well-developed nuclear  changes.

FIG. 3.

Lace-like  network  of  vacuoles  at  tissue margin  from  the  same culture  ( X 130).

FIG. 4.

Outgrowth of  normal human renal cells fixed  and stained  with  hematoxylin  and eosin. Control for cultures shown in Fig. 5-7 (×110).

FIG. 5.

A portion of  the cell outgrowth shown in Fig. 4  more highly  magnified. Hematoxylin and eosin  stain  (×300).

FIG. 6.

Outgrowth of  human  renal  cells  showing  giant cell formation and nuclear  changes  20 days after inoculation with 2 ml blood  from Case 3. Hematoxylin  and eosin  stain (×110).

FIG. 7.

A portion  of  cell outgrowth shown in Fig. 6  more  highly  magnified. Hematoxylin  and eosin  stain  ( X 300).

Certain  of  the  biologic properties of  the agents isolated from patients with measles have been definitely determined, others  in  a  preliminary  or  tentative  manner. In several instances these properties have been studied  only in  respect  to the  strain first  isolated  from the  blood  of  Case  3. Since, however,  the  other  strains,  as  far  as they  have been  examined, behave in a similar manner  it is probable  that all of  them, when  thoroughly studied,  will  exhibit  the  same  general  characteristics.

As  repeatedly  stated, agents have  been  recovered  from  both  blood and  throat washings. In three cases yielding viruses  from  one  or  another  of  these  sources fecal suspensions have likewise been examined by  the  tissue  culture  technic. In  none  was evidence for the presence of  an agent obtained. Further  examinations of  fecal  specimens are necessary before  it can  be  stated  whether  or not  the  virus  is present  in  the  intestinal excreta.

Monkey kidney is  the  only  other  tissue  employed  that  has yielded  a  growth  of  cells in  which  the  characteristic  changes  described  above  have  been definitely  observed  following  inoculation  of virus. In cultures  consisting  largely  of  monkey  renal  epithelial  cells  as  prepared  by Youngner’s modification of  Dulbecco’s technic (13)  cytopathic  changes  have  been  regularly observed  which  resemble  closely  those  produced by these agents in human renal cells as seen  in  both  fresh  and  stained  preparations. These effects followed the addition of  blood or throat washings from cases of  measles as well as infected  tissue  culture  fluids  derived  from previous  passages. Monkey  kidney  cultures may,  therefore,  be  applied  to  the  study  of these  agents in  the  same  manner  as cultures of  human  kidney. In  so  doing,  however, it  must  be  borne  in  mind  that  cytopathic effects which  superficially  resemble  those  resulting  from  infection  by  the measles  agents may possibly be induced by other viral agents present  in the monkey  kidney  tissue  (c f.  last paragraph  under  G)  or  by  unknown  factors. In a  few cultures  of  human  prepucial  tissue inoculated  with  one  of  the  measles  agents changes  resembling  those  seen  in  renal  cells were noted  in  the  epithelial  outgrowth  about certain  fragments. Additional  observations, however,  will  be  required  before  it  can  be confidently asserted that dermal epithelial cells are  specifically  attacked  by  these  viruses. In a single  experiment  no  cytopathic  manifestations  were  seen  during  a  period  of  31 days  following inoculation  of  infected  tissue culture  fluid  into cultures  of  human  embryonic  skin  and  muscle,  human  uterine  tissue or  embryonic  chick  tissue. Tests  for  the presence  of  complement  fixing  antigen  in the  fluids  removed  from  the  cultures  on  the 31st  day  were  negative. These  serologic results suggest that growth  of  the virus did not occur,  since,  as will  be  shown  subsequently, the antigen appears to develop regularly  after several  days  in  cultures  of  renal  tissue  infected with  the  virus.

Two  litters  of suckling white  mice  (1-day-old)  were  inoculated  with  infective  tissue  culture  fluid  by both  the  intraperitoneal  and  intracerebral routes. The animals remained well during an observation  period  of  21  days. Employing the same material as inoculum 0.1 ml amounts were introduced into the amniotic sac of  7-day embryonated  hen’s eggs. After 7 days  incubation  at 36°C the  amniotic  fluid  and membranes  were harvested,  ground  with  alundum and centrifuged at 1500 rpm. The supernatant  fluid  was  used  for  a  second  egg  passage which  was  carried  out  in  the  same  manner. Whereas inoculation  of  0.1 ml aliquots  of  the first  egg  passage  material  into  cultures  of monkey  renal  epithelium  was  followed  by characteristic  cytopathic  changes  on  the  8th day, the addition of  0.5 ml of  second egg passage material to such cultures failed to produce this effect. No complement fixing antigen was detected  in  the  materials  from  the  egg  passages. Although these results suggest that the virus is not  readily  adapted  to growth  in  the chick embryo,  it is evident that much  further investigation will be required  to determine  the degree  of  susceptibility of  this  host.

Serial passages of several of  the strains have been carried out with out difficulty in cultures of  human or monkey renal  cells. Employing  0.1  ml  of  the  fluid phase  as  inoculum,  10 passages  of  the  first strain  isolated  have so far been  accomplished in  human  cells  of  this  type. With  other strains fewer passages have been completed as indicated  in  Table  I.

As  yet  only  one attempt has been made to measure infectivity of  virus propagated  in tissue  culture.
In this case  fluids  and  cells  were  removed  from  15 cultures  of  human  kidney  tissue  on  the  6th day  after  inoculation  of  fluid  from  the  4th tissue culture passage of  the agent  from  Case 3. These materials were pooled and the  cells were ground with  alundum  in  the presence of the fluid. After centrifugation for  15 minutes at 2500 rpm the supernatant fluid was titrated for  infectivity  in  cultures  of  human  kidney tissues.
For  this purpose 3  cultures were each inoculated  with  0.1  ml  of  the  suspension  diluted  by  a  factor  of  10. The  endpoint  of viral activity as indicated by the highest  dilution  causing  cytopathic  changes  was  about 10-2.5. This low titer was somewhat unexpected in view of  the fact, as will be shown hereafter,  that  tissue  culture  fluids contain  sufficient  antigen  to  fix  complement  in  the  presence of  convalescent measles serum. Without more experimental data, however, it cannot be assumed  that  maximal  infectivity  titers  lie within  this range.

That  the  cytopathogenic  capacity  of  at least  one  strain  of the agents associated with measles is inhibited by serum factors developing during the course of  the  disease  has been  demonstrated  in  two experiments.
Employing 100 ID50 of  the viral suspension  mentioned  in  the  previous  paragraph  neutralization  tests  were carried  out in cultures of  monkey renal epithelial cells. Sera taken  during  the  acute  and  convalescent stages from  two of  the  cases occurring  at the boys’  school  were  stored  at  -15°C  and  inactivated  at 56°C  for 30 minutes  before  they were diluted and used  in the test. As diluent bovine  amniotic  fluid  was  employed. Dilutions  of  serum  and  virus  were  mixed  and kept at 5°C  for one hour when 0.1 ml of  each mixture  was  added  to  each  of  three  tissue cultures. In  both  tests  the  cultures  were examined  every day or every  2  days  and  the final readings were recorded on the  10th day. The results are summarized in Table 11. They indicate that significant increases in substances occurred  in  the  serum  of  both  patients  that neutralized the cytopathogenicity of  the agent isolated  from the blood of  a third  patient. In considering these  results  it is pertinent  to recall  that  agents  with  similar  characteristics have been isolated from the two patients whose convalescent  sera were shown to possess virusneutralizing  capacity.

TSBLE II.

Neutralization of  Cytopathogenic Effect of  Virus from Measles Case 3 by Convalescent Sera  from  Two  Other  Cases  of  Measles. *Readings taken  on  10th  day  after  addition  of virus.

Since it has been shown (17)  that  in  cultures  of  poliomyelitis  viruses antigens capable  of  fixing complement  in  the presence  of  specific  antibodies  appear  in  the fluid  phase,  tests  were  carried  out  to  determine  whether  or  not  the  fluids  removed from  cultures  exhibiting  cytopathic  changes induced  by  the measles  agents  might  behave in  a  similar  manner. To  this  end  the  drop method  of  Fulton and  Dumbell(18)  as modified  by  Svedmyr,  Enders  and  Holloway(17) was  employed. As  antigens  crude  undiluted fluids  have  been  used. These  were  derived from  human  or  monkey  kidney  cultures  inoculated  with  strains  isolated  from  either blood or throat washings of  Cases 2,  3  and  4. The fluids from several cultures were collected at  various  intervals  after  inoculation  of  the virus,  pooled,  centrifuged  at  1500  rpm  for 5 minutes and stored at either  5°C or -16°C. Immediately  before  use  the  fluid  was  heated at  56°C  for  30 minutes  to  remove  any anticomplementary  activity that might be present. As  control  antigens,  fluids  were  taken  from uninoculated  cultures  maintained  under  the same conditions as well as fluids from cultures of  the  agent  producing  changes  superficially similar  to  those caused by the measles agents and  which  are mentioned  below. These  materials  failed  to  fix complement  with  any  of the sera that have been examined. Acute  and convalescent  phase  measles  sera  were  inactivated  at  56°C  and  serial  dilutions  prepared as in  complement  fixation  tests  for poliomyelitis antibody. The results of  tests that have so  far  been  completed  indicate  clearly  that antibodies  develop  during  the  course  of measles  capable  of  reacting  with  an  antigen that appears in  the culture fluid after the 3rd to  the  7th  day  following  inoculation  of  the virus. From  representative  data  presented in  Table  III it  is  evident  that  the  antibody may  emerge at least  as early  as the  7th  day following the appearance of  the rash and continues to persist for at least 2  months in fairly high  titer. By  this  time,  however,  there  is some  indication  that  the  maximal  concentration has been previously attained. It is noteworthy  in  respect  to  the  possible  etiologic relationship  of  these  agents  to  measles  that antibodies  appeared  in  the  blood  of  Cases  6 and  7  which fixed complement with  the  antigens from Cases 2  and 3. The latter occurred in  a  widely  separated  area  and  at an  earlier time. A few tests have been  carried  out with measles antigen on sera from 3 adults giving a history  of  measles  during  childhood. Serum titers  of  1:2,  1:8 and  >1:16 were  recorded.

TSBLE III.

Results  of  Complement  Fixation Tests  on  Measles  Sera  Employing  as  Antigens Fluids from  Tissue  Cultures  Infected with  Agents Isolated  from  Cases  of  Measles.

* MKC = Monkey  kidney  tissue  culture  fluid  infected  with  agent  from blood  of  case  2.
†  HKC = Human  kidney  tissue  culture  fluid  infected  with  agent  from blood  of  case  3.

Filtration.

A portion  of  the pool  of virus  with  infectivity  titer  of  l0-2.5 (cf.  last paragraph  under  E)was diluted  1:5  in  beef infusion  broth  and  passed  through  a sintered glass  filter  under  a  pressure  of  45  mm  Hg.
Time of  filtration  was  10 minutes. The  capacity  of  the  filter to  retain  Serratia  marcescens was then demonstrated. The viral  filtrate was  shown to be  free of  bacteria  by addition to infusion and thioglycollate broth and blood agar  media. Five  cultures  of  monkey  renal cells were  inoculated  with  the  filtrate  (0.5-1 ml). Characteristic  cytopathic  changes were subsequently noted  in all.

Thermal stability.

The cytopathogenicity  of  one  strain  was  destroyed by heating at 65° for 30 minutes. The infectivity  of  heparinized  blood  or  throat washings  in  milk  as  tested  in  tissue  culture was preserved for at least 3 1/2  hours by refrigeration at 1°C to 5°C. Agents present in tissue culture fluids remained infective after 38 days at -15°C and after at least  35 days at about -50°C  to -60°C.

Two agents have been isolated while the present work was in progress that appear unrelated to those we have just described. The first was recovered from the throat washings of  a typical  case  of  measles  occurring  in  the  boys' school. Its  wide  cytopathogenic  range,  the character  of  the  cytopathic  changes  induced and the fact that its infectivity for tissue  cultures  was  neutralized  by  herpes  simplex  immune rabbit serum served to define its nature. A second agent was obtained from an uninoculated  culture  of  monkey  kidney  cells. The cytopathic changes it induced in the unstained preparations  could  not  be  distinguished  with confidence  from  the  viruses  isolated  from measles. But,  when  the  cells  from  infected cultures  were  fixed  and  stained,  their  effect could  be  easily  distinguished  since  the  internuclear  changes typical of  the measles agents were  not  observed. Moreover,  as  we  have already indicated, fluids from cultures infected with the agent failed to fix complement in the presence  of  convalescent  measles  serum. Obviously the possibility of  encountering such agents  in  studies with measles should  be  constantly kept in mind.

Of  the  numerous  experiments that have been reported in the past describing the successful isolation  of  the etiologic agent of  measles only those in which monkeys were employed  as  the  experimental  animal  have been consistently confirmed by other workers. Great  caution  should  therefore  be  exercised in  the  interpretation  of  any new  claims  that the virus  has  been  propagated  in  other  hosts or  systems. Accordingly,  the  results  that are  summarized  here  must  be  subjected  to the  most  critical  analysis.

The  following  facts  tend  to  support  the hypothesis that the viruses we have  described are responsible for the disease. Experimentally transmissible agents exhibiting a similar and characteristic cytopathogenic effect in cultures of  human  or  simian epithelial cells have been isolated  from either the blood or throat washings  derived  from  5  of  7  typical  cases  of measles  during  the  early  acute  phase. An agent  was  demonstrated  in  the  blood  of  4  of the  5  cases  from  which  specimens  were  obtained  and  examined  by  the  tissue  culture method. These findings would  seem to be  of especial  significance since  it  is  unlikely  that viruses unrelated  to measles would be regularly  present  in  the  circulating  blood  of  these individuals some of  whom were geographically widely  separated.

The  pathologic  changes  induced  by  the agents in  epithelial  cells in  tissue  culture  resemble,  at  least  superficially,  those  found  in certain  tissues  during  the  acute  stage  of measles. While  there  is  no  ground  for  concluding  that  the  factors in vivo are the same as those which underlie  the formation of  giant cells  and  the  nuclear  disturbances  in  vitro, the appearance of  these phenomena in cultured cells is consistent  with  the  properties  that  a priori  might  be  associated  with  the  virus  of measles.

The  emergence  of  antibodies  during  the course  of  the  disease  capable  of  suppressing the  cytopathogenic  effect  and  of  fixing  complement  in the presence of  infected  tissue culture  fluids  affords  further  evidence  for  the close  association  of  the  agents  with  measles. Obviously additional  data  to be  derived  from tests with  sera  from  a large  number  of  cases of  measles as well as other infectious diseases, especially the common exanthemata, are desirable in order to eliminate any remaining doubt concerning the specificity of  these serologic reactions. The  accumulation  of  such  data  is now  in  progress.

Although  we  have  thus  already  obtained considerable  indirect  evidence  supporting  the etiologic role of  this group of  agents in measles, 2 experiments essential in the establishment of this  relationship  remain  to  be  carried  out. These will consist in the production of  measles in the monkey and in man with tissue culture materials  after a number  of  passages  in vitro sufficient  to  eliminate  any  virus  introduced in the original inoculum. The recovery of  the virus  from  the  experimental  disease  in  these hosts should then be accomplished.

The findings just summarized support  the  presumption  that  this  group  of agents  is  composed  of  epresentatives  of  the viral  species responsible  for measles.

Eight  agents  exhibiting  the properties of  viruses have been isolated in cultures of  human  or simian renal  cells from the blood  or  throat  washings  of  five  cases  of typical  measles. Multiplication  of  the agents in  vitro  is  accompanied  by  characteristic changes in the cells. Primarily these changes consist in the formation of  syncytial giant cells wherein  the  chromatin  assumes  a  marginal position  and is replaced  centrally  by  an acidophilic  substance  of  unknown  nature. The cytopathogenic  effect  of  at  least  one  of  the agents  is  inhibited  by  convalescent  phase measles sera from other patients with measles. Antigen appears during cultivation in vitro of the  measles  agents  that reacts  specifically in complement  fixation  tests  with  convalescent phase  measles sera.

1.  Enders,  J.  F.,  Virus  and  Rickettsial  Diseases, Harvard  University  Press,  Cambridge,  Mass.,  1940, pp237-267.

2.  Van  Raooyen,  C.  E.,  and  Bodes,  A.  J.,  Virus Diseases  of  Man.  Thomas  Nelson  and  Sons,  New York,  1948,  pp226-228.

3.  Rake,  G.,  Measles  in  Viral  and  Rickettsial Infections of  Man,  ed. T .  M.  Rivers,  J.  B.  Lippincott  Co.,  Philadelphia,  1952,  pp480-481.

4.  Rake,  G.,  and  Shaffer,  M.  F.,  J.  Immunol., 1940,  v38,  177.

5.  Rake,  G.,  Shaffer,  M.  F.,  and  Jones,  H.  P., J. Inf. Dis.,  1941,  v69,  65.

6. Plotz, H., Bull. Acad. Med., 3rd Ser., 1938, v119, 598.

7.  Shaffer, M.  F.,  Rake,  G.,  Sitokes,  J.,  Jr.,  and O’Neil, G.  C.,  J.  Immund.,  1941,  v41,  241.

8.  Stokes,  J.,  Jr.,  O’Neil,  G. C.,  Shaffer,  M.  F., Rake, G.,  and Mark,  E. P.,  J. Pediat.,  1943,  v22,  1.

9.  Maris, E.  P., Rake,  G., Stokes, J.,  Jr.,  Shaffer, M.  F., and  O’Neil,  G.  C.,  ibid., 1943,  v22,  17.

10.  Shaffer,  M.  F.,  Personal  communication  to JFE.

11.  Rsobbins,  F.  C.,  Weller,  T.  H.,  and  Enders, J.  F., J.  Immunol.,  1952,  v69,  673.

12. Enders, J. F., hoe. Soc. Em. BIOL.  AND  Mm., 1953,  v82,  100.

13.  Youngner, J.  S.,  ibid., 1954,  VSS,  2012.

14.  Warthin, A.  S ,  Arch.  Path.,  1931,  v l l ,  864.

15.  Mulligan,  R.  M.,  ibid.,  1944,  v37,  61.

16. Bonenfant, J. L., Arch. Fran. de Pediatrie,  1952, v9, 497.

17.  Svedmyr, A.,  Enders, J. F., and Holloway,  A., Proc. Soc. Exp. BIOL. AND MED., 1952, v79, 296.

18. Fulton, F. and Dumbell, K. R., J. Gen. Microbiol., 1949, v3, 97. 

Received  May  16,  1954.  P.S.E.B.M.,  1954,  v86.