SUSC E‘SIBELEW @F EUMAN ANNEQN (FL) CELL LiNE TO TH§2 ECHO VIRUSES Thesis for #510 Degree of M. S. i‘i‘HCHIGM STATE UNIVERSITY Betsey Jane Kmeger 1958 .‘eia'SlS SUSCEPI‘IBILITY OF HUMAN AMNION (FL) CELL LINE TO THE ECHO VIRUSES Betsey Jane Krueger A THESIS Submitted to the College of Science and Arts Michigan {State University of Agriculture and Applied Science in partial fulfillment of the requiremts for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1958 ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. w. N. Mack, of the Department of Microbiology and Public Health, for his patient guidance and supervision throughout the entirety of this investigation. Appreciation is also extended to Dr. L. C. Ferguson, of the Department of Microbiology, and to Dr. E. S. Beneke, of the Department of Botany, for their helpful suggestions and criticisms of this thesis. Sincere thanks are also extended to Russell Krueger, to Henry Bloom, and to all others whose cooperation and encouragement made this study possible . TABLE OF CONTENTS PAGE INTRODIUCTIONOOOOOOOOOOOOOOOOOO... 1 I. The Enteric Cytopathogenic Human Orphan (ECHO) Viruses o o o o o o o c o o o 0 II. Hmman Ammion Cell Cultures o o 0-0 a o o o o «no ‘MATERIALS AND METHODS o o o o c o o o o o o o o o o o 9 I. Cleaning and Preparation of Glassware . . . o 9 II. Media o o o o o o o e o I o o o o o o e o c o 9 III. TISSUE Culture 0 o o o o o o o o o o o o c o 10 IV. Virus strains. 0 o o o o o o o o o o o o o 12 V. Monkey Kidney Tissue Culture Inoculation . o 12 VI. FL Cell Strain Inoculation . . . . . . . . . l2 HE'SU'LTSCOO-OOOOOOOOOOOOOOOOOOOO lo DISCUSSIOPIOOOOOOOOOOOOOOOOOOOOOC 2] SIWYOOODOOOOOOOOOOOOOOOOOOO 25 SELECTEDBIBLIOGRAPHY ................ 26 INTRODUCTION The purpose of this investigation was to determine the susceptibility of the human amnion (FL) cell line to the enteric cytopathogenic human orphan (ECHO) viruses. The study was undertaken with two purposes in mind: (1) to determine if the FL cell strain would be suitable for the routine isolation of enteric viruses and (2) to further characterize the ECHO viruses. The use of a continuous cell line, such as the FL strain, has several advantages over the use of a primary culture, such as monkey kidney, for virus isolation. Perhaps one of the major disadvantages in the use of primary cultures is that they often have been found to contain contaminating viruses from the source animals. Hull 23‘; 3;. (1956) and Brown (1957) have reported on spontaneously occurring simian viruses from tissue cultures of monkey kidney cells. Another advantage of the continuous cell culture is that it is generally easier to maintain and least expensive from the laboratory standpoint. Also, its use eliminates repetition of time-consuming preparation of tissues for primary cell cultures. Primary cell. cultures exhibit varying susceptibility to the same viruses due to variations in source animals while continuous cultures usually do not vary as much in msceptibility. Therefore, it can be seen that a. continuous cell line similar in susceptibility to the same viral spectrum as the prinery monkey kidney culture would be most desirable. Recently Fogh and Lund (1957) described the continuous cultivation of cells derived from a normal human semiotic membrane, which they call the FL strain. The susceptibility of the FL strain to the E13110 viruses was investigated to determine if this cell line, with en the inherent advantages of a continuous culture, would be susceptible to a. portion of the viral spectrum of primary monkey kidney cultures. I. The Enteric Cytopathogenic Human Orphan (ECHO) Viruses The discovery by Enders, Weller, and Robbins (19h9) that polio- mrelitis viruses would multiply in tissue cultures of human embryonic tissue and the subsequent report by the 8am group of men, Robbins, Enders, and Weller (1950), that polioviruses caused cell injury and death (cytopathogenio effect) in tissue cultures provided the criteria by which the presence of viruses could be recognized in 3.1.9.120 These discoveries led to the widespread use of various types of tissue cultures in all phases of poliomyelitis research. Among the different aspects of this research was the use of rhesus monkey kidney tissue cultures for screening of fecal specimens from patients with suspected poliomyelitis. It soon became evident that ammg the polioviruses that were being isolated in tissue cultures other viruses were also present. Robbins at 51... (1951) in a study on the direct isolation of virus strains from patients with non-paralytic and paralytic poliomyelitis found cytopathogenio agents that could not be classified among any of the known vimses. Melnick (1953) reported 10 agents, other than polioviruses, that were isolated from human stool specimens using monkey testicular tissue cultures. None of the ten was neutralized by poliomelitis virus antisera. Three of these agents were pathogenic for suckling mice, which would place them in the Coxsackie group of viruses, the other seven exhibited no mouse pathogenicity. In another publication, Melnick (1951:) reviewed unpublished data on the isolation of these unclassified agents by various investigators and referred to them as "orphan viruses". 1 . "n1“ fOI th- an. (I- (‘1 PT Rams-Alvarez and Sabin (19511) isolated 31 monkey kidney tissue culture cytopathogenic agents from 1566 rectal swabs of healthy children. Of these 31 agents, 5 were polioviruses, one was a Group B Coxsackie virus, and 25 belonged to a group of viruses they called the "human enteric viruses". Horstmann (1955) while surveying endemic virus infections in Egypt found 113 of 319 infants infected with som agent which exhibited cytopathogenic effects in monkey kidney tissue cultures. Fourteen of these children were excreting polioviruses, eight Coxsackie viruses, and 91 others "orphan viruses". In 1955 a comdtte consisting of G. Dalldorf, J. F. Ifinders, W. McD. Harmon, A. B. Sabin, J. T. Syverton, with J. L. Melnick as chairman, working under the auspices of the National Foundation for Infantile Paralysis, reported on various aspects of this new group of viruses (oemittee on the ECHO Viruses, 1955). The committee was formed to evaluate the significance of these new viruses. This group of viruses which had previously been called "orphan viruses" and "human enteric viruses" were now named the "enteric cytopathogenic human orphan (ECHO) viruses". The ECHO group of viruses were isolated through the use of tissue culture techniques from fecal specimens of patients with the clinical syndrome of aseptic meningitis, from healthy children in various areas of the world (the Philippine Islands, Egypt, and the United States), an! during surveys of epidemics occurring in the poliomyelitis season. The committee reported the recognition of 13 antigenically distinct WHO viruses sharing certain properties. All were isolated in monkey kidney tissue Cultures and were cytopathogenic for both simian and human cell cultures. Antisera against the three poliovirus types, Coxsackie group B and Coxsackie group A type 9 viruses did not neutralize their cytopathogenic effect on tissue cultures. They produced no disease in suckling mice nor in other small laboratory animals. The committee also reported that they were not related to other viruses recovered from the alimentary tract by tissue culture techniques. The ECHO viruses are neutralized by individual human serum and by pooled human gamma globulin. The ECHO group is further characterized in the committee's report as follows : Other studies of the ECHO viruses (more extensive for some than for others) have provided additional information. Complement-fixing antigens have been detected in the culture fluids of a number of viruses that have been tested. All the viruses tested were ether-resistant. Ultrafiltration (graiocol membrane) measurements indicated sizes for types 1, 2, and 3 betwaen 11 and 17 m. The size of type 10 is reported to be between 60 and 90 up. Plaque morphology of the ECHO viruses studied (types 1, 3, h, 5, 6, 7, and 9) is sufficiently distinctive, except for type 7 (Garnett strain), to rmit differentiation from polio virus plaques. e plaques of the ECHO viruses mentioned had irregular diffuse boundaries, and healthy cells could be found within the degenerated areas. Kidney cells of different monkey species vary in their susceptibility to the ECHO viruses. Rhesus (Macaca mulatta) and cynomolgus (M. irus) cells are susceptible to all 13 types studi'é'd. Cells from the South American capuchin (Cebus c ina) were found to be resistant to types 1, 2, , 7, 8, 9, and 11. However, they were susceptible to type 10. Cells from the African red grass military monkey (Weave Eatas), which were resistant 1701371333 0 .9 s 9 s ,and9,wereas susceptible as those from the rhesus monkey to the type 7 Garnett strain. (Comittee on the ECHO Viruses, 1955, page 1188) In 1957, the name of the Connnittee on the ECHO Viruses was changed to the Comittee on the Enteroviruses (1957) to include in its consideration not only the ECHO viruses but also the polioviruses and the Cossackie viruses, Groups A and B. The committee reported that six new ECHO antigenic types had been recognized and confirmed thus bringing the number of ECHO viruses to 19. Other investigators have reported various properties of the ECHO group since the first report by the Committee on the ECHO Viruses (1955). Lerner gt ;a_l_. (1957) reported on the susceptibility of primary human chorion cells to ECHO types 1 - it. They found that ECHO viruses 1, 3, h, 6, and 11 produced rounding of the epithelial cells while sparing many of the fibroblasts in the cultures. ECHO viruses 2, 7, 8, 9, 10, 12, 1.3, and 1h produced no cytopathic changes in the cultures and did not multiply. In comparing the behavior of ECHO types 1 - 16 in fibroblast-like aid epithelial-like mman cell strains, Stulberg, Page, and Berman (1958) found that all the ECHO types (except type 10) multiplied in the fibroblast-like cell line. Of five epithelial-like cell strains that were tested the ECHO viruses either did not grow at all or only a few types grew in the various strains. Archetti, Weston, and Wanner (1957) found that ECHO types 1 through 13, with the exception of type 1;, were adaptable to HeLa cell cultures with cytopathogenic effect to the cultures. They also reported that the material from the 5th passage in HeLa cultures gave specific reliable antigens for use in the complement fixation test. Using tissue cultures from human amniotic membranes, Lahello (1957) found that these primary cultures were more susceptible to WHO virus type 6 (1 - 2 log units higher) than monkey kidney tissue cultures. Studying the effect of hydrogen ion concentration on the cytopatho- pathogenicity of ECHO viruses, Barron and Karzon (1957) found that the cytopathogenic titer of certain ECHO viruses in monkey kidney tissue cultures was depressed or delayed when grown in the presence of a radium that developed an acid pH. The ECHO viruses have not been connected with any specific diseases at this time. However, ECHO viruses types 6 and 9 have been incriminated as the etiologic agent in cases of aseptic meningitis. Davis and Melnick (1956) reported the isolation of ECHO type 6 from 224 cases of aseptic meningitis. Karzon gt _a_1_. (1956) have also reported the isolation of ECHO type 6 from seven cases of aseptic meningitis. It was stated by Davis and Melnick (1956) that " . . . the present report of echo virus type 6 as the only agent associated with 21; cases Of the disease lends strong suppOrt to the view that this virus is one of the etiological agents of aseptic meningitis " . Rhodes arr! Beale (1957) also incriminate some of the ECHO viruses, along with Coxsackie group B viruses and other viruses, among the viral causes of aseptic meningitis. Faulkner gt 2;. (1957) isolated an agent, later identified as ECHO type 9, from stool specimens (and cerebrospinal fluid, in one instance) of 3 patients with aseptic meningtis. ECHO virus type 9 was found to be the etiological agent in an epidemic of aseptic meningitis which occurred in Western Europe (Quersin-Thiry gt 11., 1957). Partly on the basis that this agent was pathogenic for suckling mice, they proposed that ECHO virus type 9 be reclassified as a member of the Coxsackie group A viruses. (The ECHO virus type 9 isolated by Faulkner gt 3:}. also was pathogenic for suckling mice.) II. Human Amnion Cell Cultures The continuous human amnion cell strain (FL) as developed by Fogh and Lund (1957) is conposed of epithelial-libs cells. The cell line was started from a normal Inman amniotic membrane by trypsin digestion in November 1956 and has been cultivaWd in serial passage since that time. Beyond the original report of the continuous cultivation of cells from the amniotic membrane by Fogh and Lund no subsequent reports on the use of this cell line for virus propagation have appeared. However, there have been several reports on the use of primary amniotic membrane cell cultures for virus propagation. In the hope that characteristics of the primary cultures are similar to the continuous cultures the findings of several investigators are sumarized. Lahelle (1957), Weinstein (1956), Dunnebacke (195a), and Wilt (1956) have all fault! that humn amnion cells are susceptible (as reflected by cytopathogenic changes) to the three types of polioviruses. Dunnebaclee (1956) repormd that human amnion cells differed from HeLa, monkey kidney, and human fetal cells in their cellular changes when infected with the polioviruses. The virus was released approximately I48 hours later in amnion cells. Other differences concern the nucleolus and the formation of small nodules on the cell's surface. ‘ Tabemoto and Lerner (1957) found that primary human amnion cell cultures were susceptible to the adenoviruses types 1 - 8 , Group 11—9 Coxsackie virus, types Bl, B3, and BS, and one of four BZ strains of Coxsackie viruses, and herpes simplex virus. Weinstein (1956) found that Coxsaclcie type B3 produced cytopathogenic effect but Group B types 2, 3, and it did not. In addition the primary amnion cell cultures were susceptible to Coxsackie Group A type 9, herpes simplex, and western equine encephalomrelitis viruses but not influenza viruses strains A and B. Infectious bovine rhinotracheitis (Cheatham and Crandell, 1957) and measles (Milovanovic _e_t 31”., 1957) viruses have also been grown in primary human amnion cultures. Primary amnion cultures are reportedly more susceptible to polioviruses and ECHO viruses than monkey kidney cultures (Lahelle, 1957). Using 11 strains of ECHO virus type 6, Lahelle obtained titers l - 2 log units higher when the virus was propagated in amnion cell cultures than when it was grown in monkey kidney cultures. Comparing the relative usefulness of primary amnion cultures for the routine isolation of enteric viruses, Kelly (1957) found that they were not as satisfactory as a‘combination of mnloey kidney cells and suckling mice but better than HeLa or Detroit-6 cells as determined by the number of virus isolations from each system. MATERIALS AND METHOIB I. Cleaning and Preparation of Glassware All equipment (glassware, filters, pipettes, etc.) was washed in a- warm solution of Haemo-sol (Heineclce and Co., New York, N. Y.), rinsed 6 times in tap water, 6 times in distilled water, and once in glass- distilled water. The glassware was sterilized by dry heat at 300° F for three hours. Other equipment was sterilized in the autoclave at 15 lbs pressure (121° C) for 15 minutes. II. Media The nutrient medium used to maintain the monkey kidney epithelial cells was made from 10X concentrate synthetic Medium 199 (Morgan, Morton, and Parker, 1950) with 2% inactivate horse serum. The hydrogen ion concentration of the medium was adjusted to a range of 7.0 to 7.2 with 2.87: NaHCO3. This medium will be referred to as 15-199. The nutrient medium used for the cultivation of the amion cell line consisted of Eagle's (1955) basal medium (BME) with 20% inactivated human serum. For virus propagation BME with 5% inactivated calf serum was used . The cosmonauts of Eagle's basal medium and synthetic mixture 199 were obtained from Microbiological Associates, Inc., Bethesda, Maryland. Glass-distilled water was. used to make up all media. To avoid bacterial contamination 100 units of penicillin and 100 micrograms of streptomycin were added to each m1 of medium. Humn, horse, and calf sera were obtained from normal animals by allowing the blood to clot and removing the serum. The serum was centri- fuged at 2000 rpm for 10 minutes in an International Centrifuge, size 2, 10 Model V, to remove any red blood cells, inactivated by heating in a water bath at 5o° c for 30 minutes, and tested for bacterial sterility. Before routine use all sera were checked in tissue culture to determine if they were free from toxic properties and then stored at -20° C until they were used. The trypsin solution used for dispersion of the amnion cell cultures was prepared from Bacto-Trypsin 1:250 (Difco Corp. , Detroit, Michigan) in a concentration of 0.25% by weight using calcium free Hank's balanced salt solution as diluent. The solution was sterilized by Salts-filtering under negative pressure. III. Tissue Culture Monolayer cultures of rhesus monkey kidney epithelial cells, used for titration of the infectivity of the viruses, were purchased from Microbiological Associates, Inc. These were shipped by air express in 16 x 150 mm screw-cap tubes ready for use. After arrival the medium was replaced with 0.5 ml M—l99 and the tubes were incubated at 37° C for at least 21; hours before inoculation with the viruses. The original culture of the continuous amnion (FL) cell line was received from The Carver Foundation, Tuskegee Institute, Alabama. Stock cultures of the amnion cell line were cultivated in 8 ounce prescription bottles. Cultures were transferred weekly in the following manner : the nutrient fluid was removed from the cell layer and replaced by an equal volume of 0.25% trypsin solution. The trypsin solution remained on the cell layer until the cell sheet was loosened and floated in the solution. This required about 5 to 10 minutes at room temerature. With a pipette the trypsin solution, containing the cells in suspension, was transferred to a 15 ml conical centrifuge tube. The solution was ll dram into and dispelled from the pipette several times to break up clumps of cells. The cell suspension was then centrifuged at 600 rpm for 10 minutes. The supernatant fluid was removed and the cells were resuspended in 5 ml of BNE containing 20% inactivated human serum. At this time a- count of the cells was made. One part of the F1. cell suspension was mind with nine parts 0.1% crystal violet in 0.1M citric acid to facilitate counting. A hemocyto- meter was used to determine the number of cells in suspension. Calcula- tions were made according to the method presented in Dignostic Procedures _f_9_l_: Lig 2."; Rickettsial Diseases (1956). The cell suspension was then diluted to contain 100,000 cells/ml. and transferred into new culture bottles. Ten m1 of the suspension were placed in each culture bottle. At first, culture tubes (16 x 150 mm) were made using one ml. of a suspension containing 50,000 cells/ml. When it was found that a good monolayer of cells was not obtained using this concentration of cells it was increased to 75,000 cells per tube. This concentration produced a better monolayer of cells and was used thereafter. Both culture bottles and tubes were incubated at 37° C in a horizontal, stationary position. Nutrient mdium was replaced in the culture bottles every 148 - 72 hours. At the end of 5 or 6 days a layer of cells covered the side of the bottle. On the seventh day the cultures were trypsinised and transferred. The culture tubes were not disturbed for 6 days. At the end of this time the fluid was removed, the cells were washed three times with Hank's balanced salt solution and 0.5 ml fresh BME containing 5% inactivated calf serum was added to the tubes. The tubes were examined 12 under the low power of the microscope ard those tubes with good cellular growth were inoculated with viruses. IV. Virus Strains Nineteen prototype ECHO viruses were investigated. ECHO viruses types 1- 6 and 111 were obtained from the American Type Culture Collection, ECHO viruses types 7 - 13 and 16 were received from Dr. Stulberg, Child Research Center, Detroit, Michigan, and ECHO viruses types 15 and 17 - 19 were received from Dr. Wanner, University of Kansas, Kansas City, Kansas. Three prototype polioviruses, type I (Mahoney), type II (MEF-l), and type III (Saukett) were used as positive controls to infect the amnion cell cultures. All viruses were stored at -20° C. V. Monkey Kidney Tissue Culture Inoculation Titrations of the ECHO viruses were performed in the susceptible monkey kidney tissue cultures to determine the infectivity titer (TCDSO) of the viruses. Ten-fold dilutions of the individual viruses were made in M-199. Each dilution was inoculated into 3 tubes of monkey kidney cells using 0.1 ml per tube. The tubes were examined daily for 10 - 12 days after inoculation for microscopic signs of cellular degeneration produced by the virus. Media were replaced on the culture tubes every 3 or 1; days. The fifty-percent endpoints were calculated by the method of Reed and Muench (1938) and expressed as TCD50/ml. Each titration was done mice. VI. FL Cell Strain Inoculation FL cell strain cultures were inoculated with 0.1 ml per tube of undiluted or 10"1 dilutions of the respective ECHO and polio viruses. As calculated from the infectivity titers of the viruses the undiluted 13 and 10-1 dilutions contained from 103 to 108 tissue culture infecting doses per 0.1 ml. The exact inoculum used of each virus is presented in Table II. Each virus was inoculated into three tubes (0.1 ml per tube) of FL cells and observed for cytopathogenic effect. The medium in the tubes was replaced every three or four days. The medium that was removed from each set of three tubes was pooled and frozen. All medium from the respective virus inoculations were pooled separately to be used for further inoculations or for blind passages. If no cytopathogenic effect was observed blind passages were made. (See Figure I for a schematic presentation of FL cell strain inoculations with the ECHO viruses). This was done by the inoculation of 0.1 ml per tube of the pooled medium into another set of 3 FL culture tubes. Blind passages were performed to get a madam; expression of cytopathogenicity of the virus through possible adaptation to the FL cell strain oyster. At the end of the third passage in the FL cells the pooled material from the 3rd passage was subcultured into monkey kidney culture tubes to determine presence or absence -of virus. The viruses which showed no demonstrable cytopathogenic effect in monkey kidney cultures were reinoculated, using the original virus, into FL cells as above. At the end of the observation period the pooled nutrient fluid was inoculated into monkey kidney cultures to determine presence of absence of virus. It was necessary to have a control to determine if the virus could have survived in the medium for the original passage, without any multiplication occurring. Therefore, tubes containing 0.5 ml medium and no cells were inoculated with 0.1 ml per tube of the respective viruses. These tubes were incubated at 37° C for the same period of time as the FL cell culture tubes. At the end of this time 0.1 ml of Rams meme Sumo ~8an sees: TI 62330 .. flame ca 25 2893.283 83 zoESSozH £qu no; name ESQ go: n 85 2051885 nfioo .E 9." newsman an. MMEQHM NBEOZ 82H ZOHBEOOZH 20g BEE UHZMOOdenHOHHo 02 go ESQ M8502 AIIIIIOBZH zOHBSDOOZH zomb BREE ofiamoomedmoao 09ml EN Ileana II 5,3me peace as 5 fineness mmmDmH> omom BE. EH3 onadqboozH zHgm dmo Hm ho 20HB.omom H mnmdfi 18 3.30 Hm 3.. newsman m scams .Mfi a.“ mmo pecan oasomofioaopho on .- 7v poomuo awesomenemaopho .- Ti annoy asses .N roots «Homogeneous .H + I I I H ma r I I I I JOH Nd + I I I :0H 0H _. - - .. .. as a a. I I I I OOH 4n .__ I I I I mOH 9.. + I I I I OOH Nu ... I I I no." .3” + I I I I 40H OH + I I I I 00H m + I I I I 40..” m + I I I I wOH .a. a. I I I mo." 0 + I I I I m9” m + I I I I med d + I I I mg m + I I I noa m + I I I I 00H .n nice .E as .58 as an ran as sea as commons swamped .n nomad homemade m gonad owmmmdm sudsoofi menu. . In .2 5 Eu «.2 5 E0 38 as ea ammo H538 nah» onion ”858.50 HEAR—”M M8502 8,2 Ego Hm 82H mmmDme 030m #0 ZOHHfiSUOZH ho megbmmm HH am; 19 the 7 - 8 day observation period. At the end of the observation period the pooled media from each of the viruses was inoculated into another set of FL culture tubes. No cytopathogenic effect was observed for a period of 12 days. Another passage in FL cultures was made and during this third passage no cellular degeneration was caused by any of the nineteen ECHO viruses. None of the viruses in any of the three passages produced typical or observable cellular degeneration in the FL cultures. Subculturing of the pooled, third passage material into monkey kidney cultures showed that six of the ECHO viruses caused rapid cellular destruction of the cultures. ECHO viruses types 2, 3, b, 11, 16, and 19 caused lysis of the monkey kidney cells which began either on the first or second day after inoculation. Rapid lysis, such as this, would indicate a large amount of virus present. The remaining thirteen ECHO viruses did not cause any cytopathogenic effects in the monkey kidney cell cultures during the ten day observation period. Since no virus was present after 3 passages in FL cultures for thirteen of the ECHO viruses it was desirable to know if any virus could be detected after a single passage in these cells. Upon reinocula— tion of the thirteen viruses into FL cultures and subsequent subculture of the first passage material in monkey kidney cells ECHO virus types 1, h, 5, 7, 8, 9, 10, 12, 13, 114, and 17 caused cellular degeneration of the monkey kidney cultures. ECHO virus types 15 and 18 did not cause any cytopathogenic effects in the monkey kidney cell cultures during the twelve day observation period. Indicating that ECHO virus types 15 and 18 were destroyed during one passage in FL cell cultures. 1 When the ECHO viruses were incubated in medium containing no cells only ECHO virus types 7 and 1h survived the incubation period. The 2O incubated virus material of the remaining thirteen showed no activity When tested in the susceptible monkey kidney cell cultures. ECHO viruses 2», 3, 6, ll, 16, and 19, the viruses which caused cellular destruction of the monkey kidney cultures after three passages in FL cell cultures, were passed We more times in the amnion cell cultures . When the pooled material from the fifth passage was inoculated into monkey kidney cultures all six ECHO viruses caused cellular degenerat- tion of the cultures which began either on the first or second day after inoculation. Because of the dilution factor involved in five passages viral multiplication must have occurred without any apparent damage to the FL cultures. ECHO viruses 2, 3, 6, 11, 16, and 19, plus ECHO viruses types 1, 7, 9, 13, and 17, when inoculated into armion cell suspensions did not cause cellular degeneration of the cultures. The amnion cells settled onto the wall of the tubes and formed a monolayer in the same maimer as if no virus had been present. No cellular degeneration was observed in the monolayers during the ten day observation period. DISCUSSION The results indicate that the continuous human amnion (FL) cell line is refractive to the majority of the ECHO viruses. Thirteen of the viruses, except types 15 and 18, existed through one passage but not through three passages in the FL cell line. The presence of ECHO virus types 15 and 18 could not be detected after only one passage in FL cultures. Six of the viruses, types 2, 3, 6, 11, 16, and 19, were maintained with viral multiplication through five passages in FL cultures. None of the ECHO viruses, but all of the polioviruses, produced cellular degeneration in the cultures. The titration values obtained in this study fall within the range of results obtained by other investigators (Table I). The findings that the amion cell cultures did not produce couplets monolayers and that the cultures did not survive for as long as monkey kidney cultures are disadvantages in the use of the continuous amnion cell line. The less complete monolayer obtained in amnion cell cultures makes it more difficult to observe cellular degeneration. In the case of the polioviruses it did not seem to be a deterring factor because the cellular degeneration was complete, with few of the cells remaining attached to the wall of the culture tube after a period of six days. If cellular degeneration had occurred to a lesser degree visual observation of cellular degeneration would have been more difficult. In the attempt to enhance cellular degeneration by exposure of the cell suspension to the virus there was still no observation of cytopatho- genic changes, which would suggest that viral multiplication was occurring without cellular destruction. The work of Stulberg 33 gl_. (1958) suggests that epithelial-like cell 22 strains would be most refractive to support of ECHO virus multiplication and cytopathogenicity. They found that of five epithelial cell lines (they did not use the FL cell strain) infected with ECHO viruses none of the lines underwent cytopathogenic changes, except for an occasional ECHO type causing cellular degeneration. For the most part, the epithelial cell lines were refractive to the ECHO viruses tested. On the basis of this observation one might have suspected that the amnion cell line would also be refractive to the ECHO viruses. It does not hold true, however, that all epithelial cell lines are non-supporters of ECHO virus multiplication. The susceptible monkey kidney cultures are composed of epithelial cells and they do support viral multiplication of the ECHO viruses, with cytopathogenic changes occurring in the cultures. The present work would indicate that the human amnion cell line is not suitable for routine ECHO virus isolation. A suitable cell line would be one in which viral activity was associated with observable cellular changes occurring in the culture. Since the FL line does not fulfill this requirement it would therefore not be suitable. The F1. line might possibly be used to separate the ECHO viruses from the polioviruses in such the same way as a selective medium might be used to separate bacterial groups, as suggested by Deinhardt and Henle (1957). Both the ECHO viruses and the polioviruses can be isolated in monkey kidney cultures from clinical specimens. However, the cytopathogenic effects of these viruses in no way distinguish them from each other. Identification is usually made through serum neutralization tests. Therefore, if it was possible to separate these groups of vimses on the basis of cell line susceptibility, identifica- tion would be faster and easier. Even though there is evidence that 23 some of the ECHO's do multiply in the amnion cell line none show cytopathogenic effects while the polioviruses do. Before such a system could be employed, however, considerably more work would be necessary to determine the complete spectrum of viruses that cause cytopathogenic changes in FL cell cultures. ' (The present work does not necessarily establish that the human amnion (FL) cell line is not susceptible to cellular destruction by the ECHO viruses. There is the possibility that the FL cell line used in this laboratory is different from that of the original cell line. It is in the realm of possibility that the cells underwent modification or mutation during the time they have been carried in serial passage. It would be necessary to repeat the work in an amnion (FL) cell line maintained by another laboratory before a final conclusion could be drawn. On the basis of amnion cell culture inoculation it would appear that the ECHO viruses could be divided into two groups:, one group consisting of thirteen viruses that do not multiply nor cause cellular degeneration of the cultures and a second group of six viruses that apparently multiply without causing cellular degeneration. The significance of this grouping cannot be estimated at this time. Lerner gt a. (1957) in investigating the susceptibility of human chorion cells to ECHO viruses types 1 - 11; found that the viruses also fell into W0 groups on the basis of cytopathic changes in the cultures. They found that ECHO viruses types 1,. 3, h, 6, am 11 produced cytopatho- genic changes in the chorion cell cultures while types 2, 7, 8, 9, 10, 12, 13, and 11; did not. The groupings of Lerner 219 51;. and the groupings determined in this study, while not identical, show some overlapping. These characterizations, coupled with future investigations, might eventually lead to the recognition of divisions among the viruses of the ECHO group. 2h SUMMARY The susceptibility of the continuous human amnion (FL) cell line to 19 prototype ECHO viruses was determined. None of the ECHO viruses caused cellular degeneration of FL cultures even when cell suspensions were inoculated with the viruses. Monkey kidney cell cultures were used to determine if the viruses had multiplied without causing observable cytopathogenic effects in the FL cultures. ECHO virus types 2, 3, 6, ll, 16, and 19 caused no cytopathogenic effect through five passages in FL cultures but inoculation of monkey kidney cultures showed that viral multiplication had occurred. After three passages in FL cultures no virus could be detected in monkey kidney cell cultures for ECHO virus types 1, h, 5, 7, 8, 9, 10, 12, 13, 1h, 15, 17, and 18. Virus was detected after only one passage in FL cells for all these viruses except 15 and 18. It was concluded that the ECHO viruses could be divided into two groups on the basis of FL cell inoculations. One group consisting of thirteen viruses that do not multiply nor cause cellular degeneration ‘ of FL cultures and a second group of six viruses that apparently multiply without causing cellular degeneration. SELECTED BIBLIOGRAPHY American Public Health Association. 1956. Dignostic Procedures £93 Virus and Rickettsiagl Diseases. 2nd Ed. A.P.H.A. Publications Office, ”New York City. p. 116. 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Association of ECHO virus type 6 with aseptic meningitis. Proc. Soc. Exp. Biol. Med. 92: 839-8173. Deinhardt, F. and Henle, G. 1957. Studies on the viral spectra of tissue culture lines of imman cells. J. Immmo. 19: 60-67. Dunnebacke, T. H. 1956. Cytopathic changes associated with polionwelitis infections in human annion cells. Virology, g: 811-819. Eagle, H. 1955. Nutrition needs of mammalial cells in tissue culture. Science, £23: 501-501.. Enders, J. F., Weller, T. H., and Robbins, F. C. 1919. Cultivation of the Lansing strain of poliomelitis virus in cultures of various human embryonic tissues. Science, 192: 85-87. Faulkner, R. S., MacLead, A. J. and vanRooyen, C. E. 1957. virus meningitis -- seven cases in one family. Can. Med. Assoc. J. 17: 1139-14113 . Fogh, J. and Lund, R. O. 1957. Continuous cultivation of epithelial cell strain (FL) from human amniotic mexnbram. Proc. Soc. Exp. Biol. ’I'bde 2g: 532-537e 27 Horstmann, D. M. 1955. Endemic virus infections in Emt: isolation of poliomyelitis viruses and other tissue culture pathogenic agents from infants. Federation Proc. Q: 1.66. Hull, R. »N., Minner, J. R. and Smith, J. W. 1956. New viral agents recovered from tissue cultures of monkey kidney cells. Am. J. Hyg. Q: 2074-215. Karzon, 13. T., Barron, A. L., Winkelstein, W., Jr., and Cohen, S. 1956. Isolation of ECHO virus type 6 during an outbreak of seasonal aseptic Mtise J. Am. Had. ASSOC. 29-2-3 1298-1303. Kelly, S. 1957. Enteric virus isolations from sewage. Acts Medics Scand. £52: 63. Lahelle, o. 1957. _ Multiplication of polio and ECHO viruses in tissue culture prepared from human amniotic membranes. Acta Pathol. et Microbiol. Scand. 92: h36-hhh. Lerner, A. H., Takemoto, K. H., and Shelokov, A. 1957. Human chorion cells: cultivation and susceptibility to viruses. Proc. Soc. Exp. B101. Med. 2g: 76-80. Melnick, J. L. 1953. 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SUSCEPTIBILITY OF HUMAN AMNION (FL) CELL LINE TO THE EHO VIRUSES Betsey Jane Krueger AN ABSTRACT Submitted to the College of Science and Arts Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 195 8 Betsey Jane Krueger ABSTRACT The susceptibility of the human amnion (FL) cell line to 19 strains of enteric cytopathogenic human orphan (ECHO) viruses was investigated to determine if this tissue culture strain would be suitable for routine ECHO virus isolation and to further characterize the ECHO viruses. The cultivation of the FL cell line is described and the advantages of a.continuous cell line are discussed. Culture tubes containing FL cells were inoculated with ECHO viruses types 1 - l9 and polioviruses types I, II, and III to determine if the viruses cause observable cellular degeneration (cytopathogenic changes) in the cultures. None of the ECHO viruses, but all of the polioviruses, caused cytopathogenic changes in the cultures. Monkey kidney epithelial cell cultures were used to detect presence or absence of ECHO viruses in the nutrient medium after passage of the viruses in the FL cultures. 311:0: the ECHO viruses, types 2, 3, 6, ll, 16, and 19, were maintained through five passages in.FL cultures. The remaining thirteen.ECHO viruses, except types 15 and 13, were present after one passage but not after three passages in the FL cell line. On this basis the ECHO viruses could be divided into two groups: one group consisting of thirteen viruses that neither multiply nor cause cellular degeneration and a second group of six viruses that apparently multiply without causing observable cellular degeneration. The study indicates that the human.amnion (FL) cell line is not suitable for routine ECHO virus isolation. The possibility of using the cell line in identification of the ECHO viruses and the polioviruses is discussed. uLUu‘. ddflzg r I: Ain‘t}: . .1 .i l flu in h I . V. a a...