126 646 THS CULT“ ANGIE ' ‘ C3? é "iE‘EC‘s’ECfic E’R‘C “ifi‘i'ifi "JERUS .~ m' w: HM: .« ., WW! 3*???" I»?! N;ri.{: i‘tijl £11.3‘L U 1:\.»’$‘..:L nus”...- w s ; mask {can ‘30 was cf 3;. $. as: 11mg: 1 '\ I wztiT—w L “'2”; ‘. )2. 52 JHESIS LIBRARY Michigan Stan University CULTIVATION OF INFECTIOUS BRONCHITIS VIRUS IN CHICKEN EMBRYO KIDNEY CELLS By MARTHA P. SPRING A THESIS Submitted to the College of Science and Arts of 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 1960 MUM Fur/~90 ACKNOWLEDGMENTS I wish to extend my appreciation to Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, for his consideration, encouragement and guidance through- out the investigation and the preparation of this manuscript. My appreciation is also extended to Dr. H. J. Stafseth, Professor (Emeritus), of Microbiology and Public Health, for his encouragement, inspiration, stimulus and faith in my abilities to undertake graduate study. Sincere thanks are expressed to Mrs. D. W. Church for her cooperation and technical assistance. INTRODUCTION. TABLE OF CONTENTS REVIEW OF LITERATURE. MATERIAL, METHODS AND RESULTS DISCUSSION. . SUMMARY . . . BIBLIOGRAPHY. O 10 28 35 37 TABLE 1. 2. LIST OF TABLES PAGE Neutralization tests in cell culture. . . . . . . 20 Adsorption of virus on chicken embryo kidney Cells 0 O O O 0 O O O O O O O 0 O O O O O O O O O 22 Effect of storage on chicken embryo kidney cell- adapted IBV I O O O O 0 O O O O O O O O O O O O O 27 LIST OF FIGURES FIGURE PAGE 1. Normal chicken embryo kidney cells (x 100). . . . l4 2. Chicken embryo kidney cells infected with the 54th cell passage of chicken embryo kidney cell- adapted infectious bronchitis virus (x 100) . . . l4 3. Normal chicken embryo kidney cells (x 200). . . . 15 4. Chicken embryo kidney cells infected with the 54th cell passage of chicken embryo kidney. . . . 15 5. Infectivity of chicken embryo kidney cell-adapted infectious bronchitis virus at different passages as titrated by cell and embryo response . . . . . l7 6. Procedure for study of the adsorption of chicken embryo kidney cell-adapted virus to the cells and release from the cells. . . . . . . . . . . . . . 24 7. Multiplication cycle of chicken embryo kidney cell-adapted infectious bronchitis virus. . . . . 25 INTRODUCTION Previous studies of animal viruses and their relationship to the host cell prompted investigation of infectious bronchitis virus in this same category. The purpose of this research was to study the virus-cell interrelation of infectious bronchitis virus and chicken embryo kidney cells. The objective was the adaptation of a chicken embryo-adapted strain of infectious bronchitis virus to chicken embryo kidney cells and to determine if any cyto— pathic effect of the virus could be used for study of the multiplication cycle. REVIEW OF LITERATURE Infectious bronchitis, a specific respiratory disease of chickens, is caused by a virus with an average diameter of 60 to 100 mp (Reagan gt_§l., 1948; Reagan 33 ‘al., 1950; Reagan and Brueckner, 1952). Infectious bronchitis virus (IBV) can be propa- gated readily in chicken embryos by various routes of inoc- ulation (Beaudette and Hudson, 1937). On primary isolation of the virus in chicken embryos, gross pathological lesions such as "curling and stunting" of the embryos, excessive urates in the kidneys, and clubbing of the down are observed. Mortality of em- bryos is variable (Beaudette g£_§l., 1957; Cunningham, 1952; Cunningham and Stuart, 1947; Cunningham and Jones, 1955; Delaplane and Stuart, 1941; Fabricant, 1949; Fabri— cant, 1956; Loomis g£_§l., 1950). Serial passage increases the virulence of the virus for embryos, but decreases the virulence for chickens. The criterion of complete adaptation of the virus to em- bryos is the ability of the virus to kill all embryos within 24 to 36 hours. Embryo-adapted virus is nonimmunogenic but is specifically neutralized by anti-infectious bron- chitis serum (Beaudette g£_§;., 1957; Delaplane and Stuart, 1941; Fabricant, 1951; Page, 1950; Page, 1954). 2 Fahey and Crawley (1956) cultivated the Connaught Laboratories vaccine strain R and the Beaudette embryo- adapted strain of IBV in stationary flask cultures of frag- ments of minced chicken embryo kidney, liver, and chorio— allantoic membrane (CAM) and in monkey kidney cells. The virus multiplied in the tissue cultures as determined by chicken embryo infectivity tests, but cytopathic effects (CPE) could not be observed in the infected cultures. These strains did not infect mouse liver cells or Hela cells (Davis, 1956, cited by Buthala and Mathews, 1957). Buthala and Mathews (1957) were not successful in prOpagating the 170th embryo passage of a strain of IBV in monolayers of chicken embryo kidney cells (CEKC). Chomaik gt_al. (1958) reported that CEKC may be infected with the Beaudette strain as shown by CPE. The Massachusetts and Connecticut strains did not cause infec— tion. The 10th CEKC passage of the Beaudette strain caused CPE of a few chicken embryo fibroblasts. The CPE of the virus became more extensive with further passages in fibro- blasts. The Beaudette strain can be serially passed in whole and minced CAM suspended in Hank's balanced salt solution (B88) and in Morgan's medium 199 (Cunningham and Spring, unpublished data; Ferguson, 1958; Hanks, 1949; Ozawa, 1959). Growth was evident in the 12th passage which represented a dilution of the virus far in excess of the titer of the original seed virus. This indicated that an adaptive process was necessary for tissue culture as well as for chicken embryo cultures. Mortality of embryos was used as the criterion of infectivity for the CAM-passaged virus (Ferguson, 1958). Hitchner and White (1955) also used mortality of embryos as the criterion for infectivity in growth-curve studies of the Beaudette strain. Any embryo showing one or more of the criteria of curling and stunting, clubbed down, or excessive urates was recorded as infected by the Connaught R strain. The log phase for the Beaudette strain began four hours postinoculation. The maximum concentration was reached at 12 hours. The Connaught R strain entered the log phase in six hours, and the maximum concentration of the virus was attained within 24 to 50 hours. Ackermann g£_§1. (1954) demonstrated multiplica- tion of poliomyelitis virus, Saukett strain, Type III, using Hela cell monolayers. Tube cultures containing 105 Hela cells were inoculated with one ml of tissue culture fluid containing 107’5 tissue culture infective doses (TCID50)' The cultures were incubated at 57 C for one hour. The inoculum was removed, and the culture was washed five times with maintenance medium. After one m1 of nutrient medium per tube was added the cultures were incubated at 57 C. At hourly intervals, 0.1 m1 portions of fluid were removed for titration in other tubes of Hela cell cultures. Each portion of fluid that was removed was replaced by the same volume of fresh medium. The latent period was nearly four hours. The release of virus during the next five to six hours was at an exponential rate. The maximum yield was reached within 10 to 11 hours. A single sequence of in- fection was produced in Hela cells by use of a massive inoculum of poliomyelitis virus. The CPE of the virus was used as the criterion of infectivity. The growth curve was similar to a single sequence of infection produced in cultures with influenza or Western equine encephalomyelitis (WEE) virus. Lack of synchronization of infection of in- dividual cells was indicated by the differences in the rate at which cytOpathic alterations developed in the cells. Fahey and Crawley (1956) found in growth curve studies using stationary flask tissue cultures of minced CAM that the adsorption period for the Beaudette strain, sixth tissue culture passage was from four to eight hours. The latent period varied from 12 to 18 hours. The maximum titer was obtained in approximately 40 hours, as contrasted to 12 hours in chicken embryos. The multiplication cycle, using trypsinized monkey kidney cells, revealed that no fresh virus appeared for eight to ten hours. No CPE was evident. Growth curve studies by Ozawa (1959) with the Beaudette strain grown in a suspension of CAM in BSS showed the following phases of the multiplication cycle: (1) a variable lag phase of eight hours; (2) a logarithmic phase during the next 52 hours; (3) a primary decline phase over the following 60 hours; (4) a stationary phase of 72 hours; (5) a secondary decline phase of 48 hours. The Beaudette strain cultivated in CEKC was used by Chomiak gthl. (1958) in growth-phase studies. Titra- tions in the cell cultures were used with CPE as the in- fectivity response of the virus. The virus disappeared from the extracellular fluid within four hours, reappeared in 16 hours, and reached a maximum titer at 48 hours, where it remained for 24 hours and then declined. When titrated in chicken embryos, the titer of the CEKC-adapted virus was 103 higher than when titrated in CEKC. No statement was made as to the cell passage of virus used, but it may be assumed that it was in one of the first 10 CEKC passages. In addition to the microscopic examination of infected cell cultures for CPE as an indication of viral infectivity, certain animal viruses may be assayed by the plaque technic. Wright and Sagik (1958) reported plaque forma— tion by the Beaudette strain in CEKC. Plaques were apparent in 16 to 18 hours. After a three-day incubation period, the plaques were three to four mm. in diameter and could be seen without the aid of the microscope before staining. Titers obtained by the plaque count (PFP) and by embryo infectivity showed a ratio of four when it was assumed from the Poisson distribution that one embryo lethal dose50 equals 0.7 virus particle. Dulbecco and Vogt (1954) investigated the rate of adsorption of WEE virus on whole chicken embryo cells by the plaque technic. One-step growth curves were deter- mined in cell suspensions and on cell layers. The latent period was shorter with the cell layer and varied between two and three and one half hours, followed by an initial exponential rise reaching a maximum after six to eight hours. Rubin g§_§1. (1955) used the plaque technic to study the intracellular appearance and release of WEE virus in suspensions of infected chicken embryo fibroblasts. No intracellular virus was found during the first hour after adsorption, indicating that the virus was non-infectious after entering the susceptible cell. The first progeny of the virus was detected in the cells between one and two hours after infection and increased in amount exponentially during the next three hours. The released virus in the extracellular fluid increased at the same rate, but ex- ceeded the intracellular virus by a factor of 20 during the period of the exponential increase. The authors con- cluded that a virus particle could be released from the cell within one minute after infection. Howes and Melnick (1957) inoculated monolayers of monkey kidney cells with poliomyelitis virus, Brunhilde type, for studies of the maturation and release of the virus in a single growth cycle. At various intervals the virus in the cell monolayer, cell-associated virus (CAV), and "free" virus in the medium was assayed by the plaque technic. An accurate picture of virus maturation in this system was obtained by considering that the total virus was composed of CAV plus "free" virus. The virus was pro- duced at a slow rate, and only 50 per cent of the total amount of virus appeared within four hours and 48 minutes to six hours and 12 minutes. This is in contrast to WEE virus in chicken embryo fibroblasts where the virus was released rapidly, and "free" virus was considered to be an approximation of the "total" virus. Levine and Sagik (1956) demonstrated a one-step growth curve of Newcastle disease virus (NDV) on whole chicken embryo monolayers by the plaque technic. The latent period of three to four hours was followed by a log period which lasted from four to eight hours. Rubin and Franklin's (1957) experiments assaying NDV by the plaque technic using chicken embryo lung cells determined the velocity constant for adsorption and multi- plication of the virus. "Free" virus and CAV were equal in amount during the period of the exponential increase of virus. The apparent release time was calculated to be about 80 minutes. Howes (1959 A) studied the maturation and release of poliomyelitis virus, Type I, in a suspended cell popu- lation of monkey kidney cells using the plaque technic for estimating the total virus and "free" virus. The amount of "free" virus appearing during the maturation phase with suspended cells was more than that in cell mono— layers, but the growth cycles were essentially the same (Howes and Melnick, 1957). The rate of release of the virus was much less than the rate of maturation. A large proportion of the virus yield accumulated during matura- tion within the cell. The maturation phase began four hours after the virus entered the cell. The virus release began within 50 to 60 minutes after the beginning of matur- ation. Howes's (1959 B) work on poliomyelitis virus, Type I, from a single Hela cell, contributed information on the variation in time when the first mature virus was detected. A single cell released virus during one hour. or less. In multiple cell populations, the maturation and release phases of the growth cycle in individual cells overlapped. In a single cell, these phases were separated by an intracellular retention phase which varied from a few minutes to an hour. MATERIALS, METHODS AND RESULTS Preparation of Chicken Embryo Kidnengells Primary cultures of CEKC were used throughout the study. The kidneys from l6-day—old chicken embryos were removed aseptically and washed in Hank's B58 in a Petri dish. The kidneys were minced finely into pieces about 0.5 to 1.0 mm}, which were then thoroughly washed in BSS. The pieces were transferred to a 250 ml, fluted Erlenmyer flask containing a "Teflon" covered magnet, and 100 m1 of Seitz—filtered 0.25 per cent trypsin at pH 8.4 pre-warmed to room temperature. The flask was placed over a "Magnestir," and trypsinization was performed at room temperature for one hour. The cell suSpension was filtered through eight layers of cheesecloth and centrifuged at 1500 rpm (457 x G) for five minutes in an International PR-l centrifuge at four 0. The supernatant fluid was discarded, and the packed cells were resuspended in BSS. This procedure of centri- fugation and washing of the cells was repeated three times. The packed cells from the last washing were diluted 1:400 in a growth medium consisting of 0.5 per cent lactalbumin hydrolysate in B88, 10 per cent bovine serum, 100 units of penicillin and 0.1 mg. of streptomycin. A 1:400 dilution IO ll of cells averaged about 950,000 to 1,000,000 cells per ml as determined with a hemacytometer. Leighton tubes, 16 x 125 mm, were seeded with 1 m1 of the cell suspension per tube, sealed with white stoppers, and incubated at 57 C in the horizontal position. It was essential that the Leighton tubes be clean without traces of cleaning compound or without old cell cultures adhering to the walls of the tube. The tubes were cleaned in acid cleaning solution, rinsed eight times in tap water, and 12 times in distilled water. At 24 hours when the monolayer of cells was formed, the growth medium was replaced with 1 ml of a maintenance medium consisting of 0.5 per cent lactalbumin hydrolysate in B88 and two per cent bovine serum. The monolayer was then inoculated with 0.2 ml per tube of the Beaudette embryo-adapted strain, North Central Infectious Bronchitis Virus Repository, Code 42. This strain was chosen because it has the ability to kill chicken embryos within 56 hours post-inoculation via the allantoic cavity, which would serve as a means of assay of viral infectivity of the extracellular fluid from chicken embryo kidney cells. A definite routine was established and followed throughout the experiments. Trypsinized CEK primary cells were prepared and seeded. The growth medium was replaced by maintenance medium and the culture was inoculated 24 to 28 hours after seeding. 12 Micros00pic observations of the infected mono- layer were recorded 48 hours post-inoculation. The extra— cellular fluid was decanted from the tubes and pooled. The pooled sample was distributed in screw cap vials, 5 ml per vial, and stored at -60 C. Five days later the virus- infected fluid was thawed and was used as inoculum on new primary cells. Adaptation of IBV to Chicken Embryo Kidney Cells The first passage of IBV in CEKC was made by inoculating each of three tubes with 0.2 ml of virus-in— fected allantoic fluid containing 106 embryo infective doses per 0.1 ml. Microscopic examination of the cells 24 hours after inoculation revealed no apparent CPE pro- duced by the virus. The extracellular fluid was harvested, pooled, and used to inoculate three more tubes. 0n the second passage, CPE was evident 48 hours after inoculation. The cell monolayer was not uniformly affected, but it was evident that infection of the cells had occurred. The affected cells were rounded and clumped to- gether. The periphery of the cells was dense, and the cytoplasm was clear without inclusion bodies or granules. Degeneration and necrosis were the over-all picture. In some areas of the monolayer, the dead cells had sloughed 13 from the wall of the tube. There was a tendency for the dead cells to aggregate (figures 1, 2, 5 and 4). By the fifth passage, the virus was well adapted to propagation in CEKC. The entire monolayer showed evidence of viral infection. The virus was serially passaged 55 times. Titration of Virus in Chicken Embgyo Kidney Cells The fifth cell culture passage of the virus was used for the first titration of viral infectivity using CPE as the positive response. Serial ten-fold dilutions of the virus were pre- pared using as the diluent 4.5 ml of B85 containing 0.5 per cent lactalbumin hydrolysate. The virus was thawed and 0.5 ml was transferred to the first tube with a 2 m1 serological pipette. With another 2 ml pipette, the con- tents of the tube were mixed by aspirating and expelling the mixture from the pipette 20 times, and then transfer- ring 0.5 ml to the next tube. This was continued until all dilutions were made. The virus dilutions were kept in an ice bath to prevent inactivation of the virus at room temperature. Each dilution of virus was used to in- oculate three tubes of cell culture, 0.2 ml per tube, which were incubated at 57 C for 48 hours when microscopic ex- aminations were made. Figure 1. Normal chicken embryo kidney cells (x 100). ”338 "--*‘"“ ‘ 'r 5:- ' “1:22. . "‘ 'L g: I.“ :43‘15214 Figure 2. Chicken embryo kidney cells infected with the 54th cell passage of chicken embryo kidney cell-adapted in- fectious bronchitis virus (x 100). 15 .2 - ‘9 ‘- 220'“. :l‘.’ " hfi'fii'a... 'l 1 I .'\“"‘?:'£' ‘ _ ‘ J a 1 \ 1r . .1 (471(93- * HE&‘§£MM$3 ’. - , a) ‘ ~ .‘ '. Ari...“ - ~‘71flasfggkfi :’ ‘o-_ a o . ‘ ‘ q. INS “" .: . . Figure 5. Normal chicken embryo kidney cells (x 200). Figure 4. Chicken embryo kidney cells infected with the 54th cell passage of chicken embryo kidney cell-adapted in- fectious bronchitis virus (x 200). tl‘l‘ll!‘l‘|’ ‘l!.lllul[ll.(l11llllllll' lilillflrlflur'l illal-.nl|!"| lll.‘ 16 The endpoint of viral infectivity was considered to be the highest dilution of the virus in which CPE was present in at least two of the three tubes. The titer of the virus was the reciprocal of the end point dilution. The titer of the fifth passage of the virus was 103 /0.2 ml in cell culture at least but 105 /0.2 ml in chicken embryos. As the virus became adapted to CEKC in subsequent passages, the titer was higher when assayed in cells than in embryos. At the 55th passage, the titer in CEKC was 109 or higher, while in chicken embryos the titer was only 105. As shown in figure 5 there was a general increase of viral infectivity related to cell culture passage as compared to a rather constant level as determined by ti- tration in chicken embryos. Neutralization Tests Decreasing virus—constant serum method Serial tenfold dilutions of the CEKC-passaged virus were prepared using the procedure previously described. Undiluted, immune and normal chicken sera were filtered through Swinney Seitz filters and inactivated at 56 C for 50 minutes. One part of the serum, 0.5 ml, was mixed with one part of the respective virus dilutions, and 0.2 m1 of the serum-virus mixture was used to inoculate each of three l7 momyQSm soxofino ca soapmnpfla N mHHmo hmsuflx ohnnam GmMOHso ma cospmapfia . mmsommmm chansm can Hamo an cmpmnpfia mm mommmmmm psmnmmwfin Pm maths mapasozonm m50fipommcH empgmsanaamo Assess cashew ewaoano so spa>apommeH .m mnsmHa ma