THE (5% GF SERIALL‘! PROPAGAIED AFEECAN GEE’EEN MQNKEY {(EENEY QELLSE EN THEE a'fiT'ECTEQN Q? A, LA’IENT NEWCAS‘E'EE B~E$EA$E WRUS m CHICKEN EMERYCW Tina from Hie Dar-ea on? me. D. MECHEGAN STATE UNEYERSETY Robert Bruce Lacey 1968 I'H F513 f, LI B RAR Y Michigan State University This is to certify that the thesis entitled THE USE OF SERIALLY PROPAGATED AFRICAN GREEN MONKEY KIDNEY CELLS IN THE DETECTION OF A LATENT NEWCASTLE DISEASE VIRUS IN CHICKEN EMBRYOS presented by ROBERT BRUCE LACEY has been accepted towards fulfillment of the requirements for PhoDo degree in MicrObiOIOgy ,/7 ///( "7 [gown] A; Major professor Date September 19, 1968 0-169 .. rwk Wm; ..4 —HO..A ABSTRACT THE USE OF SERIALLY PROPAGATED AFRICAN GREEN MONKEY KIDNEY CELLS IN THE DETECTION OF A LATENT NEWCASTLE DISEASE VIRUS IN CHICKEN EMBRYOS By Robert Bruce Lacey The initial aims of a research project are not always realized. The preliminary efforts of this investigation were directed towards adapting infectious bronchitis virus to a non-avian system. The several mammalian cells employed, including H.Ep.#2, KB, FL, AV-3, L-929, HEK, and BS-C-l, did not support the replication of IBV. Alteration of the electrostatic charges on the surfaces of the virus and the host cell through the use of a maintenance medium containing an excess of divalent cations apparently did not induce attachment, penetration, and replication of IBV within mammalian cells. An absence of compatible receptor sites on either the virus or the mamma- lian cell probably accounts for the inability of the virus to attach to, and subsequently multiply in these cells. During attempts to adapt IBV to serially propagated BS-C-l cells, a cytopathic effect was observed in tube cultures of these cells inoculated with an allantoic fluid sample of IBV strain 42. The virus which was eventually isolated from certain allantoic fluid samples of strain 42 was identified as Newcastle disease virus with character- istics very closely resembling those of the GB strain. The methods used to verify its identity were serum neutralization, hemagglutina- tion, hemagglutination inhibition, immunodiffusion, electron Robert Bruce Lacey microsc0py, and comparisons of certain of its other prOperties with those of known NDV. The original source of the NDV contaminant was unknown. While examining several samples of IBV for the presence of NDV, however, a sample of strain 42, dated March 1, 1961, was found to contain NDV. Also, a sample of supposedly "normal allantoic fluid", dated November 9, 1966, also contained NDV. The presence of NDV in a sample of "normal allantoic fluid" suggests the presence of a sub-clinical NDV infection in the flock from which eggs are obtained for research at Michigan State University. Infectious bronchitis virus interferes with the replication of NDV in a system in which both viruses can replicate, e.g., the chicken embryo. By introducing certain contaminated samples of IBV into a mammalian system, the latent NDV is free to produce characteristic cytopathic effect. THE USE OF SERIALLY PROPAGATED AFRICAN GREEN MONKEY KIDNEY CELLS IN THE DETECTION OF A LATENT NEWCASTLE DISEASE VIRUS IN CHICKEN EMBRYOS By Robert Bruce Lacey A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1968 DEDICATION Because of your avid dedication to the acquisition of scientific knowledge, your composure in the face of frustration, your willing- ness to aid others in their personal research, and your sincere friend- ship, this thesis is lovingly and most respectfully dedicated to: MRS. MARTHA P. "PAT" SPRING ACKNOWLEDGMENTS The author wishes to thank Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, and the other faculty members of the Department for their friendship and guidance throughout this study. Special thanks are extended to Dr. Keyvan Nazerian, U.S.D.A. Regional Poultry Research Laboratory, East Lansing, Michigan, for his preparation of the electron micrographs. The moral support and counsel of my fellow coworkers, especially Mr. Mark F. Stinski and Mrs. Martha P. Spring, have often been needed and always greatly appreciated. By no means least, the patience and love of a long-suffering wife and family has served as a constant source of inspiration. This study was supported in part by funds supplied by the Michigan Agricultural Experiment Station. ii TABLE 93 CONTENTS INTRODUCTION. . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . Latent viruses . . . . . . . . . . . Avian embryos. . . . . . . . . . . African green monkey kidney cells Viral interference . . . . . . . . . . MATERIALS AND METHODS. . . . . . . . . . . . Viruses. . . . . . . . . Chicken embryo kidney cell culture. . . . . Plaque assay . . . . . . . . . . . . Preparation of seed viruses . . . . . Normal allantoic fluid. . . . . . Attempts to propagate IBV in mammalian cells African green monkey kidney cell culture. . . . Preparation of antisera . . . . . . . Serum neutralization tests . . . . . . . . Erythrocytes . . . . . . . . . . . . . Hemagglutination, hemagglutination inhibition tests Immunodiffusion . . . . . . . . . . . . Thermal sensitivity. . . . . . . . . . Ether sensitivity . . . . . . . . . . pH stability . . . . . . . . . . . . Electron microscopy. . . . . . . .. . . . Sensitivity of BS-C-l cells for the detection of a contaminating virus . . . . . . . Viral interference studies . . . . . Sterility tests . . . . . . . . . . . RESULTS . . . .. . . . . . . . . . . . . Attempts to adapt IBV to mammalian cell culture . Initial isolation of the NDV-X contaminant . . . Identification of the NDV-X contaminant . . . Serum neutralization tests . . . . . Possible source of NDV- X . . . . . . Hemagglutination, hemagglutination inhibition tests Immunodiffusion . . . . . . . . . . . . Thermal sensitivity of viral hemagglutinins. iii 20 Page O‘J—‘J-‘N N O) 10 10 11 11 12 13 15 15 15 16 17 17 18 18 19 21 . 22 22 . 22 31 31 33 33 37 . 38 Ether sensitivity of NDV-X . . . . . . . . pH stability of NDV- X. . . . . . . Sensitivity of 38- C- 1 cells for the detection of NDV Growth of NDV- X in chicken embryo kidney cell cultures. Viral interference . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . iv Table LIST SE TABLES Neutralization indices of chicken antisera against IBV-42 , NDV-GB, and NDV-X0 o o o o o o o o o o o 32 Hemagglutination of NDV-GB and NDV isolates with erythrocytes of various species. . . . . . . . . . 35 Hemagglutination inhibition titers of various antisera against NDV-GB, NDV isolates, and influenza PR-8.. . . . 36 Thermal stability of viral hemagglutinins. . . . . . . 38 Ether sensitivity of NDV-X. . . . . . . . . . . . 38 pH stability of NDV-X.. . . . . . . . . . . . . 39 Serum neutralization and hemagglutination inhibition tests demonstrating interference by IBV on the replication of NDV-X in chicken embryos.. . . . . . . . . . . 42 LIST 9g FIGURES Figure 1. Uninoculated BS-C-l cells . . . . . . . . . . . 2. BS-C-l cells, 24 hours after inoculation with NDV-X. . . 3. BS-C-l cells, 48 hours after inoculation with NDV-X. 4. Electron micrograph of negatively stained NDV-X from allantoic fluid. . . . . . . . . . . 5. Electron micrograph of negatively stained NDV-X from allantoic fluid showing the rupture of the intact viral particle with the release of the NP antigen . 6. Thin sections of 38-0-1 cells infected with NDV-X . . . 7. Thin sections of BS-C-l cells infected with NDV-X 8. Immunodiffusion reactions . . . . . . . . . . 9. Plaques produced on CEKC by NDV-X 5 days after infection . vi 26 27 28 29 30 37 40 INTRODUCTION Laboratory animals, chicken embryos, and cell cultures used for the study of viruses should be free of adventitious biological agents. Such ideal conditions are not always available, and the presence of contaminants in either the host system or in the inoculum itself can often yield complicating and undesirable results. The initial efforts in this investigation were directed towards adapting infectious bronchitis virus (IBV) to non-avian cells. During the course of these studies a strain of Newcastle disease virus (NDV) was detected as a latent contaminant present in certain allantoic fluid seed stocks of IBV. The present study is concerned with the latency of NDV in chicken embryos and cell cultures, and the isolation, identification, and par- tial characterization of this virus. LITERATURE REVIEW Latent viruses Twenty years ago (10) most researchers were convinced that chicken embryos were free of adventitious agents. As research techniques became more SOphisticated, biological experiments invol- ving the use of chicken embryos became more complex, leading in part to the unmasking of numerous latent biologic agents. Studies on NDV as a latent contaminant are well documented. Newcastle disease virus has been isolated from freshly laid eggs and from ovarian tissues (8, 40, 59); yolk sacs of 4-day-old chicks, infertile eggs (23); and dead embryos and infertile eggs during an outbreak of the disease (33). Fontaine et a1. (24) detected latent NDV in chicken embryo cells. Uninoculated fibroblastic cell cultures from apparently normal embryos degenera- ted in approximately 8 days due to NDV. The virus could not be demonstrated by either hemagglutination or by infectivity for chicken embryos during the time the cultures were microscopically normal, which indicated that the virus may have been present in a latent form. The virus could be detected only after the cells began to degenerate. It was proposed that transovarian infection occurred during or after a clinically inapparent Newcastle disease infection. The only evidence of such an infection may be a drOp in egg production and/or hatchability. Other latent viruses have been isolated from both chicken embryos and animal cell cultures. One of the most widely investi- gated latent viruses has been the chicken embryo lethal orphan (CELO) virus. This virus has been extensively studied in both chicken embryos and avian cell culture (1, 2, 17, 63, 64). The virus could be isolated from cell cultures prepared from hatched embryos inocu- lated with CELO virus (64). Ablashi et a1. (2) studied the effect of a latent CELO virus infection on the development of NDV and influenza virus. Titers of infectivity and hemagglutination of both viruses were reduced in the presence of CELO virus. Hwang et a1. (38, 39), in studies on avian encephalomyelitis (AE) virus, isolated and partially characterized an unknown virus present in chicken embryos. It was first assumed that this virus was the etiological agent of avian encephalomyelitis (38), but subsequent serum neutralization tests indicated that it was not antigenically related to AR (39). Burmester et a1. (15) reported on the presence of a virus recovered from avian lymphomas which was unrelated to avian lymphomatosis and eventually identified as Gal-virus, an avian adeno-like virus. A latent, heat resistant, ether and chloroform resistant adeno- 1ike virus isolated from chicken kidney cell cultures by Burke et a1. (13) was found to be the cause of spontaneous degeneration of un- inoculated cell cultures. The definite problem of distinguishing be- tween envolving latent viruses and viruses being studied intentionally was discussed at length. Hinze (32) isolated a herpes-like virus from kidney cell cultures of cottontail rabbits. The virus was not antigenically related to known herpesviruses, and produced a latent infection only in the cottontail rabbit. A characteristic cytOpathic effect (CPE) was produced, how- ever, in cultures of cells from animals of various Species. A similar phenomenon was reported by Clark et a1. (18) who isolated a herpes-like virus causing a latent infection in the green iguana. Four transmissible viral agents have been recovered from the feces of asymptomatic dogs by Binn et a1. (11). These viruses were heat, ether and chloroform resistant, measured 18- to 22 my in diameter, and produced CPE in a continuous dog epithelial cell line. Avian embryos The chicken embryo is a unique biological medium, and its use in many virus laboratories is Standard procedure. The developmental changes which take place during the 21 day incubation period provide a variety of biological substrates to which viruses and rickettsiae are variously adapted (52). Chicken embryos possess certain advantages, such as size, low cost, and inability of the embryo to produce antibody. The main disadvantage to their use is the ovarian transmission of certain viruses, including avian lymphomatosis, avian encephalomyelitis, and Newcastle disease virus (19). Some of these viruses are easily recognized by embryo mortality, etc., but others remain latent and serve as contaminants. African green monkey kidney cells A cell line, designated BS-C-l, was initiated from kidney cell cultures of CercOpithicus aethi0ps by HOppS et a1. (35, 36). Cells were originally prOpagated in Medium 199 (47) containing 20% fetal bovine serum and 0.1% yeastolate (Difco). More rapid cell replica- tion occurred when McCoy's 5A medium and 20% dialyzed fetal bovine serum was employed (4). According to Hopps (35), no latent viruses have been isolated from BS-C-l cells. Recently, however, a pleomorphic, heat and ether resistant RNA virus has been identified as the causative agent of an illness in laboratory workers involved with the processing of African green monkey kidneys for cell culture. Thirty cases were re- ported with 7 deaths (44). The absence Of latent simian viruses, especially SV-40, is im- portant to investigators who use monkey kidney cell cultures. Simian virus 40, or vacuolating virus, is a common contaminant of Rhesus monkey kidney cell cultures, but produces no readily identifiable cytOpathic changes. However, SV-40 does produce pathognomonic cytOplasmic vacuolation in African green monkey kidney cells (35). Studies conducted by Schmidt et a1. (56) indicated that BS-C-l cells were susceptible to infection by a wide spectrum of viruses, including poliovirus types 1 andIL certain Coxackie A and B viruses, and many ECHO viruses. The BS-C-l cells did not support the replica- tion of parainfluenza virus types 1 and 3, influenza A and B viruses, and adenovirus types 3, 4, and 7 (35). Throughout serial passage of BS-C-l cells their chromosomal characteristics varied considerably, but without effect on viral replication. The cells remained diploid, i.e., 60 chromosomes, until approximately the 40th passage. After 50 passages, the majority of the cells contained 59 chromosomes and the remainder contained 58 (35). Despite variation in the number of chromosomes, no "trans- formation" has been observed in BS-C-l cells (16). The number of BS-C-l cells in culture doubled in 72 hours. The majority of the cells in early passages of the line were: epithelial, but some fibroblastic and multinucleated cells were present in all cultures. A gradual change in appearance of the cells began after about 75 sub- cultures. The line then assumed the characteristics of established cell lines, in that practically all cells were polygonal in shape. Fibroblastic cells were rarely observed at this time (35). Viral interference Interference implies that superinfecting viral particles are prevented from entering and multiplying a previously infected cell (55). Viral interference was first described in 1935 (51). In this instance, a neurotropic strain of yellow fever virus protected monkeys experimentally infected with a lethal viscerotropic form. Extensive reviews of viral interference have been published by Henle (31) and Schlesinger (54, 55). Interference involving IBV and other avian viruses has been well documented. Heat inactivated IBV interfered with the replication of unheated IBV (25). Similar results, using heated and unheated NDV, were reported by Bang (6). The interference exerted by IBV on the replication of NDV has been reported by Beaudette (9) and Hanson et a1. (28). Infectious bronchitis virus interfered with the normal course of NDV infection in 8-week-old chickens simultaneously infected with both IBV and NDV (7, 27). Experiments to study interference between IBV and NDV were con- ducted in chicken embryos by Raggi et a1. (49). An excess of IBV consistently interfered with the replication of NDV. The time between introduction of both viruses was less important than the relative amounts of each virus administered. It was postulated that the inter— ference occurred either on, or within, the susceptible cells. However, NDV failed to replicate even though it had been introduced 4 hours prior to the addition of IBV to the test system. Newcastle disease virus should have adsorbed to the cells of the chorioallantoic membrane within 4 hours, suggesting that receptor site competition was not involved, and that the interference was intracellular, rather than extracellular. Interferon did not play an apparent role. MATERIALS AND METHODS Viruses The Massachusetts, Beaudette, and Connecticut strains of IBV, identified by the Michigan State University repository code as 41, 42, and 46, respectively, were used in the initial phases of this study. Strain 41 was in the 5th chicken embryo passage, and the titer was approximately 107°2 embryo infective dosesso (EIDSO) per ml. Strain 42 has been through numerous chicken embryo passages. The exact number is unknown but is estimated to be several hundred. The virus is completely embryo-adapted and is avirulent for the chicken. The titer was approximately 107'5 embryo lethal dose350 (ELDSO) per ml. Strain 42 adapted to chicken embryo kidney cells (CEKC) by Spring (57) was also used, and was in the 112th passage. The code number was IBV-42-112C. Strain 46 was isolated by Jungherr (41) from a commercial vaccine and was in the 7th chicken embryo passage with a titer of approximately 107°0 EID50 per ml. Seed stocks of viruses 41 and 46 were lyophilized repository cultures. The seed stock for strain 42 was a frozen allantoic fluid sample, while IBV-42-llZC was a frozen cell culture harvest. The latent virus, subsequently identified as NDV, will be referred to as "NDV-X" under the apprOpriate sections in which special techniques were employed for its identification. The velogenic strain of NDV, designated NDV-GB, was used for corollary investigations. Unless otherwise specified, all viruses, etc., were stored at -62 C until used. Chicken embryo kidney cell cultures The procedure for cell culture was similar to that described by Cunningham (21, 22). The kidneys were removed aseptically from 16- to 18 day-old chicken embryos and rinsed several times in Hank's balanced salt solution (HBSS) containing phenol red. Kidney tissues were minced into 1- to 2 mm pieces and washed free of connective tissue, blood clots, and other debris. The tissues were transferred to a sterile 500 m1 fluted Erlenmeyer flask containing a Teflon covered magnetic stirring bar. Ten ml of trypsin, 0.25% in HBSS, pH 8.0, were added for each pair of kidneys and the contents of the flask were agitated by means of a magnetic stirrer for 1 hour at room temperature. The trypsinized cells were further clarified by pouring the mixture through 2 layers of sterile cheese cloth. The cells were centrifuged at 200 x g for 5- to 10 minutes at 4 C. The trypsin solution was decanted and the cells were resuspended in HBSS. Two cycles of centrifugation and rinsing followed. The packed cells were suSpended in growth medium, 100 ml for each 1 m1 of packed cells, consisting of Medium 199 fortified with vitamins and amino acids of Eagle's basal medium, 2 mM L-glutamine, 0.1% sodium bicarbonate, 100 units of penicillin, 100 pg dihydro-streptomycin, and 50 units of Mycostatin (Squibb) per ml. Following suspension of the cells in the growth medium, 5% newborn calf serum was added to complete 10 the cultural fluid. The final concentration of cells, based on hemocytometer counts, was approximately 1 x 107 cells per ml. Four m1 of the cell suspension were dispensed into a sterile plastic tissue culture petri dish, 15 mm x 60 mm, (Falcon Plastics). The cells were incubated at 37 C in an atmosphere of 80- to 85% relative humidity and 8% COZ. Confluent monolayers were usually obtained in 48- to 72 hours. Plaque assay ApprOpriate serial dilutions of virus were prepared in HBSS without phenol red. Five per cent newborn calf serum was added to the diluent to act as a viral stabilizer. The growth medium of the cells was decanted and the cells were washed once with 3- to 4 m1 of HBSS. The HBSS was decanted and 0.5 ml of each virus dilution was added to each of 4 plates. The virus was adsorbed to the cells for 90 minutes in the 002 incubator. The inoculum was removed and the cells were overlayed with 4 m1 of cultural medium containing 0.9% Special Agar-Nobles (Difco). After 3- to 4 days of incubation at 37 C, 0.5 ml of 0.1% neutral red in 0.01 M phosphate buffer, pH 7.0, was added. The plates were incubated for 1 hour at 37 0, followed by 1 hour at 4 C. The plaques were counted and the titer of the virus was expressed in plaque forming units (PFU) per ml. Preparation of seed viruses Strains 41, 42 and 46 were propagated in the allantoic cavity of lO-day-old chicken embryos following inoculation of approximately ll 105 ED50 per embryo. Strain 42 was harvested from dead embryos approximately 36 hours postinoculation, while strains 41 and 46 were harvested from embryos living at 60- to 72 hours. All embryos were chilled at 4 C for at least 4 hours prior to the collection of virus. The allantoic fluids were pooled and stored in screw cap vials. Strain 42-113C was prOpagated in CEKC. After the cells were washed with 3 m1 HBSS, IBV-42-112C, approximately 106 PFU per 0.5 ml, were deposited on the cells. Virus was adsorbed for 1 hour at 37 C. The inoculum was decanted and 4 ml of cultural medium was added. Virus was collected approximately 36 hours postinoculation, or whenever the CPE was maximal. All samples were pooled and stored in Screw cap vials. Strains NDV-GB and NDV-X, prOpagated in chicken embryos, were harvested from embryos dead at approximately 40 hours postinoculation. In addition, NDV-X was grown in BS-C-l cells and collected 48 hours after infection, or at the time of maximum CPE. Normal Allantoic fluids Allantoic fluid was collected from normal l3-day-old embryos. Attempts to prOpagate IBV in mammalian cells Numerous attempts were made to adapt strains 41, 42, and 46 to mammalian cell cultures. Cell lines employed included the human epidermoid carcinoma of the larynx (H.Ep.#2), human epidermoid carcinoma of the pharynx (KB), two varieties of normal human amnion 12 (FL and AV-3), mouse fibroblasts (L-929), human embryonic kidney (HEX), and African green monkey kidney (BS-C-l). Only one cell line was employed in an experiment at any given time in order to prevent possible cross contamination of cells. Based on ease of handling, prolonged maintenance capabilities, and relative freedom from adventitious agents, the BS-C-l line was selected for further investigations. Initial studies were performed using tube cultures of BS-C-l cells. The cultural medium was decanted from the cells which were then washed once with HBSS. After the HBSS was decanted, 0.2 m1 of either undiluted allantoic fluid virus or cell culture virus was added to each tube. Following a 30 minute adsorption period, 1.8 m1 of maintenance medium was added to each tube. The cultures were incubated at 37 C and examined daily for CPE. African green monkey kidney cell culture Cells were routinely grown and maintained in milk dilution bottles. The growth medium most commonly used was Medium 199 in modified Earle's salts, 2 mM L-glutamine, 0.1% yeast extract, 20% fetal bovine serum, 100 units of penicillin, 100 ug streptomycin, and 100 units of tylosin tartrate (GIBCO anti-PPLO) per ml. Under these conditions a confluent monolayer of cells was obtained in 7- to 9 days. If more rapid growth was required a medium consisting of McCoy's 5A plus 20% fetal bovine serum was employed (Grand Island Biological Company). With this medium, a complete monolayer was 13 obtained within 5- to 7 days. In most cases, a maintenance medium consisting of Medium 199 in modified Earle's salts as described above was used, except the serum content was reduced to 1- to 2%. Under these conditions, BS-C-l cells could be maintained for over 3 weeks without detectible cellular degeneration. An alternative "high magnesium" maintenance medium was employed in certain studies on virus adaptation and isolation. This medium was identical to that described above except for the addition of 30 times the normal magnesium ion complement (5.072 g MgClz ° 6H20 per liter). Serial passage of BS-C-l cells was performed as follows: After a confluent monolayer of cells had formed, the cultural medium was removed and the cells were washed with 10 ml of phosphate buffered saline solution (PBS) without Ca++ or Mg++ ions. (3). The PBS was decanted and approximately 5 m1 of trypsin, 0.25% in GIBCO solution A (Grand Island Biological Company), prewarmed to 37 C, was added to the cells. After 2 minutes the trypsin was decanted. The bottles were inverted and incubated for 20- to 30 minutes at 37 C until the cells became detached from the glass surface. Twice the original amount of growth medium, minus serum, was added and the cells were resuspended by vigorous agitation. Fetal bovine serum was then added to a final concentration of 20% and 15 ml of cells were dis- pensed into bottles. Preparation of antisera Antisera against strains 41 and 46 were produced in 10-week-old 14 Single Comb White Leghorn chickens. After obtaining pre-infection sera, the birds were inoculated intranasally with 0.2 ml of virus, approximately 2 x 106 EIDSO. Twelve birds were used for each antigen. Eight weeks later the birds were reinoculated, and then 3 weeks later they were exsanguinated by cardiocentesis. Blood was collected in milk dilution bottles and allowed to clot at room temp- erature for 3 hours. Serum was then decanted, pooled, and centri- fuged at 600 x g for 10 minutes. Following centrifugation, the sera were passed through a Krueger filter, placed in screw cap vials, inactivated at 56 C for 30 minutes, quick-frozen in a dry ice-alcohol bath, and stored at -62 C. Antisera against strain 41 and NDV-X were produced in New Zealand albino rabbits. A mixture of 50 ml of undiluted allantoic fluid virus and 5 ml of a 10% A1C13 solution was adjusted to pH 7.0 with 5 N NaOH and incubated overnight at 4 C to insure complete flocculation. The fluid was gently agitated and a total of 7 ml was injected into each of 4 rabbits: 2.5 m1 intramuscularly into each rear leg, and 2.0 ml subcutaneously in the neck. Fourteen days later, each rabbit was given an additional intramuscular injection of 2.5 ml per rear leg. Two weeks later, each rabbit was injected intraperitoneally with 2.0 ml of allantoic fluid virus which had not been alum-treated. Seven days after the final inoculation, sera were collected, pooled, heat inactivated, sterilized by filtration, and stored. Sera from normal, uninoculated rabbits were processed identically 15 and served as controls. Serum neutralization tests Neutralization tests were performed using 9- to ll-day-old chicken embryos. Serial ten-fold dilutions of the viruses were prepared in either nutrient broth or tryptose phOSphate broth diluent. Three-tenths m1 of each virus dilution was mixed with 0.3 ml of antiserum. The virus controls consisted of 0.3 ml of diluted virus combined with 0.3 ml of diluent. The serum-virus mixtures and virus controls were incubated at 4 C for 1 hour. Five embryos per dilution were then inoculated with 0.1 ml of each mixture via the allantoic cavity. The embryos were candled twice daily for 5 days, and the neutralization indices of the sera were determined (50). Erythrocytes Blood for hemagglutination tests was obtained from Single Comb White Leghorn cockerels, and transferred to tubes containing 1 m1 of a 2% sodium citrate solution for each 5 ml of blood. The erythrocytes were suspended in several volumes of HA buffer (Difco) and centrifuged at 1000 rpm for 5 minutes. Washing and centrifuga- tion was repeated until the buffer was clear. After the last wash, the buffer was removed and the packed cells were stored at 4 C for as long as 4 days. Cow, sheep, horse, dog, and rabbit erythrocytes, processed as above, were also used in certain tests. Hemagglutination, hemagglutination inhibition tests The procedure of Cunningham (21) was used. From an initial 1/5 16 dilution of the virus in HA buffer, two-fold serial dilutions to 1/2560 were prepared. Each virus dilution, 0.25 ml, was transferred to a series of 12 mm x 75 mm test tubes. To these, 0.25 ml of HA buffer and 0.25 ml of a 0.5% suspension of erythrocytes in HA buffer was added. The tubes were agitated briefly and incubated at room temperature for 1 hour. The hemagglutination (HA) titer was expressed as the reciprocal of the highest dilution of the virus in which hemagglutination occurred. The erythrocyte control consisted of 0.5 ml HA buffer and 0.25 ml of the erythrocyte suspension. The procedure for hemagglutination inhibition tests was identical except that 0.25 ml of antiserum, diluted 1/5 or 1/10, was used in place of the HA buffer. The serum titer was eXpressed as the reciprocal of the lowest dilution of the virus in which hemagglutina- tion was inhibited. The hemagglutination inhibition (HI) titer of the serum was determined by dividing the HA titer by the serum titer, and multiplying the result by the dilution of the serum employed. Immunodiffusion Fifty ml of 0.3 M phosphate buffer, pH 7.1, containing 1.5% Ionagar No. 2 (Consolidated Laboratories, Inc.) and 50 ml of 16% NaCl solution were autoclaved separately. After cooling to 60 C the two solutions were combined. Ten ml of the diffusion medium were spread onto clean, grease-free, 3% inch x 4 inch cover glasses. Plastic petri dishes, 15 mm x 60 mm, were also used, and in this case 5 ml of the diffusion medium was added per dish. The plates were aged 1- to 2 days in a humid chamber at room temperature. 15]-, l7 Reagent wells were punched in the agar with a 6 mm diameter cork borer. The inter-well distance was 6 mm. The wells were filled with approximately 0.05 ml of reagents 3 times at 24 hours intervals. The plates were incubated for 5 days at room temperature in a humid atmosphere. At the end of the incubation period, the plates were dialyzed for 3 days in 6-inch petri dishes in several changes of 8% NaCl in 0.15 M phos- . 1 phate buffer, pH 7.1. The plates were then rinsed in distilled water for 5- to 10 minutes, covered with moistened filter paper, and dried at 37 C. The filter paper was then carefully removed and the plates ‘14.)... ‘ were stained with triple stain (20) for 15 minutes. Any unbound stain was removed from the plates by repeated rinsing in a 3% acetic acid solution. The plates were again air-dried at 37 C and examined. Thermal sensitivity One ml samples of allantoic fluid NDV-X diluted 1/1 in tryptose phosphate broth were diSpensed into 1 dram vials and placed in a water bath at 56 C for 30, 60, 90, and 120 minutes, reSpectively. Samples were then removed, cooled in an ice bath, and tested for hemagglutina- tion with chicken erythrocytes. Controls consisted of unheated virus held for 120 minutes in an ice bath and subsequently tested for hemagglutination. Ether sensitivity Ether sensitivity of NDV-X was determined by the method of Andrews and Horstmann (5). To 1.2 ml of allantoic fluid virus, 0.3 ml of diethyl ether was added. The mixture was thoroughly agitated and then 18 incubated at 4 C for 18 hours. Excess ether was removed by bubbling dry nitrogen through the mixture, and the resulting ether-free fluid was passed through 0.22 u Millipore filter. Serial ten-fold dilutions of the ether-treated virus were prepared in nutrient broth and tested for infectivity in chicken embryos. Controls consisted of untreated virus maintained at 4 C for 18 hours, diluted as above, and inoculated into chicken embryos. BE stability A modification of Stinski's procedure was used (58). One ml of undiluted extracellular NDV-X from BS-C-l cells, approximately 106 tissue culture infective dosesso (TCIDSO) per ml, was combined with 9 ml of either glycine-H01 buffer, pH 3.0, or glycine-NaOH buffer, pH 11.0. After 30 minutes at room temperature, the buffers were diluted 1/10 in HBSS plus 2% fetal bovine serum, pH 7.0. The mixture was then at pH 7.0. Serial ten-fold dilutions of each solution were prepared in HBSS and 0.2 m1 of each was used as inoculum for each of 5 tubes of 38-0-1 cells. After an adsorption period of 30 minutes, 1.8 m1 of maintenance medium was added to each tube. The cells were incubated at 37 C and examined daily. Controls consisted Of both uninoculated cells and cells in- oculated with non-treated virus. Electron microscopy Electron micrographs were kindly prepared by Dr. Keyvan Nazerian, U.S.D.A. Regional Poultry Research Laboratory, East Lansing, Michigan. ‘93.: 19 When CPE was present in approximately 50% of the infected BS-C-l cells they were removed from the culture bottle with a rubber Spatula and fixed for 2 hours in 1% osmium tetroxide in Zetterqvist buffer (66). The cells were rinsed in the buffer for 5 minutes and then dehydrated in a series of graded ethyl alcohols. The dehydrated cells were embedded in epoxy resin (Epon 812, Shell Oil Company), and thin sections were prepared 4 days later using a Porter-Blum MT-Z ultra- microtome equipped with a glass knife. The sections were stained with uranyl acetate and lead citrate (37, 42), mounted on grids, then coated with carbon, and examined at 80 kv in a Siemens Elmiskop 1A microscope. For negatively stained preparations, allantoic fluid samples of NDV-X were centrifuged at 12,000 x g for 30 minutes to remove gross cellular debris. The clarified allantoic fluid was then centrifuged at 74,000 x g for 1 hour. The virus pellet was resuSpended in 0.5 ml distilled water, and one drop each of the virus preparation, 2% phosphotungstic acid, and 0.01% sucrose solution were mixed. One small drOp of this preparation was placed on a Formvar-carbon coated grid and examined at 80 kv. Sensitivity of BS-C-l cells for the detection.g£ a contaminating xi_ru_s African green monkey kidney cells were tested for their ability to detect low multiplicities of infection of viruses known to replicate within this system, either alone or when combined with high concentra- tions of a virus which does not replicate in these cells. 20 The cultural fluid from monolayers of BS-C-l cells was decanted and the cells were washed once with HBSS. Newcastle disease virus strain X and IBV strain 42 were used as the test viruses, either alone or in combination. The cells were inoculated with 0.5 ml portions of NDV-X containing approximately 101 ELDSO, and/or IBV strain 42 contain- ing approximately 106 ELDSO' After an adsorption period of 1 hour at 37 C, 4.5 ml of maintenance medium was added to each flask and the cultures were incubated at 37 C and observed daily. Viral interference Studies EXperiments were performed to determine if an interference phenomenon existed between IBV Strain 42, tested and found free of NDV, and NDV-X isolated from supposedly pure stocks of strain 42 allantoic fluid virus. Approximately 106 ELD50 of strain 42 allantoic fluid virus and 101 ELD50 of NDV—X were combined. Three-tenths ml of the mixture was diSpensed into each of 3 sterile test tubes containing 0.3 m1 of either anti-IBV chicken serum, anti-NDV-GB chicken serum, or nutrient broth. After incubation at 4 C for 1 hour, 0.1 m1 of each mixture was inoculated into chicken embryos, 3 embryos per mixture. Infectious allantoic fluids were harvested from dead embryos approx- imately 48 hours postinoculation. Neutralization tests, using chicken anti-IBV serum and chicken anti-NDV serum, were then performed with the allantoic fluid harvests. Hemagglutinationéind hemagglutination inhibition tests, using the above sera, were also performed. I \I’lll1vll I]! .‘.| 21 Sterility tests Routine bacteriological sterility tests were performed, as well as tests for the presence of myCOplasma. All tests were negative. RESULTS Attempts to adapt IBV.£2 mammalian cell culture All efforts to induce replication of the 3 repository strains of infectious bronchitis virus in serially propagated mammalian cell cultures were unsuccessful, based on CPE and "blind passages." Other allantoic fluid samples of IBV from different laboratories were also tested for their ability to replicate in mammalian cells. The same results were obtained. Initial isolation of the NDV-X contaminant During attempts to adapt IBV to serially propagated mammalian cell cultures, cellular degeneration was noted in BS-C-l cells maintained on "high magnesium" Medium 199 following inoculation with an allantoic fluid sample of strain 42. The CPE closely resembled the type produced by strain 42 in CEKC. However, the rate of cellular destruction of the BS-C-l cells was Slower than the rate of destruction previously observed in CEKC infected with strain 42. Evidence of viral-induced cellular degeneration of 38-0-1 cells did not appear until 48 hours postinoculation, when syncytial cells, containing 8- to 12 nuclei, were seen. By 72 hours, the syncytia had ruptured, and there was necrosis of approximately 50% of the cells. At 96 hours, all cells were destroyed. Control cultures were normal. A single passage of NDV-X from BS-C-l cells into chicken embryos 22 23 and then back into cells produced higher virus titers, approximately 107- to 108 TCID50 per m1, and more rapid development of CPE. Syncytia were now present within 12- to 24 hours, and the cells were completely destroyed within 48- to 72 hours. The use of a ”high magnesium" maintenance medium was discontinued, Since further studies indicated that NDV-X could replicate under normal ionic conditions. The original sample of strain 42 usedas inoculum had been pre- pared in chicken embryos from randomly selected samples of virus. Fig. l. Uninoculated BS-C-l cells. 24 Fig. 2. BS-C-l cells, 24 hours after inoculation with NDV-X . 25 Fig. 3. BS-C-l cells, 48 hours after inoculation with NDV-X. 26 Fig. 4. Electron micrograph of negatively stained NDV-X from allantoic fluid (X 80,000) 27 ‘ .___ .2- .. Fig. 5. ‘Eaét Electron micrograph of negatively stained NDV-X from allantoic fluid, showing the rupture of the intact viral particle with the release of the NP antigen. (X 80,000) 28 Fig. 6. Thin sections of BS-C-l cells infected with NDV-X (X 69,000) 29 Fig. 7. Thin sections of BS-C-l cells infected with NDV-X. (X 69,000) 30 31 Identification of the NDV—X contaminant The contaminating NDV-X, isolated from supposedly pure strain 42, was eventually identified as a velogenic strain of NDV whose character- istics closely resembled those of NDV-GB. The methods used to confirm identification of the virus included serum neutralization tests, hemagglutination inhibition, immunodiffusion, and comparisons of cer- tain properties of NDV-X with those of known NDV-GB. The 4th passage of NDV-X in chicken embryos, approximately 109 ELD50 per ml, was used for most of the subsequent studies, unless otherwise specified. Serum neutralization tests Results of preliminary neutralization tests posed some real problems in attempts to identify NDV-X. It was first assumed that the virus was IBV, or perhaps an altered form of it. A neutralization test using specific, high titer chicken anti-IBV antiserum was performed, but neutralization did not occur. A chicken antiserum against NDV, prepared previously by other investiga- tors in this laboratory, was next used but it neutralized strain 42 as well as NDV-X. Samples of both rabbit and chicken NDV antisera, prepared against the mesogenic NJ-Roakin NDV strain, were received from the NDV reposi- tory at the University of Wisconsin, Madison, Wisconsin, and tested. No significant neutralization of NDV-X occurred. A standard reference antiserum against NDV-GB was next received and tested for its ability to neutralize NDV-X. This chicken anti- serum neutralized NDV-X, but did not neutralize strain 42. (Table 1.) Nm .mozunao coxowno aw voauomwom mummy k 0.0 0.m 0.0 0.m m.0 xu>02 0.0 0.0 n.m 0.m «.0 00n>02 0.0 0.0 m.0 , n.¢ 0.0 N¢n>00 A.na30 A.na30 Bowen 00 :meom A.0.m.zV A.0.m.zV mow0> HNEuoc >02 Hucm >02 Hucw >02 “and 0¢u>0H Huam *wuomwuam coxowso mo AoH omwnv mmowwcw GOMumeamuuooz .x->az can .mo->oz .Ns->mH natanmn naaaaucn emanate mo aaoanna coauanaanauaaz .0 manna 33 Possible source of NQ!;X Because the origin of NDV-X was unknown, it was necessary to re- examine all allantoic fluid samples of strain 42 for the possible pre- sence of NDV as a contaminant. After extensive investigation, a sample of strain 42, dated March 1, 1961, and stored at -62 C since that time, was found to contain NDV with properties nearly identical to those of both NDV-X and NDV-GB. During all investigations, samples of supposedly normal allantoic fluid were used as controls on BS-C-l cells. One sample, dated November 9, 1966, and stored at -62 0 since that time, produced CPE. This sample was designated "lethal normal allantoic fluid" (LNAF). Evidence of CPE in BS-C-l cells inoculated with LNAF appeared 96 hours after inoculation, and was well advanced 24 hours later. Cell fluids were collected and passaged twice in chicken embryos. The allantoic fluid harvest from embryos dead at approximately 40 hours was designated BS-C-l LNAF. Chicken anti-NDV-GB serum neutralized infectivity of BS-C-l LNAF in chicken embryos, inhibited hemagglutination of chicken erythrocytes, and produced lines of serologic identity in immunodiffusion tests. Hemagglutination, hemagglutination inhibition tests Agglutination of NDV-X was positive with cow, horse, and chicken erythrocytes, and negative with sheep, dog, and rabbit erythrocytes. When tested with chicken erythrocytes, BS-C-l LNAF caused agglutination. Specific anti NDV-GB chicken serum inhibited hemagglutination of both viruses. Partial hemolysis of chicken erythrocytes was noted in the lower dilutions of all viruses tested. 34 All controls were normal. (Tables 2, 3). Table 2. Hemagglutination of NDV-GB and NDV isolates with erythrocytes of various species. Erythrocytes cow Sheep horse dog rabbit chicken NDV-GB + - + - - + NDV-X + - + - - + BS-C-l LNAF N.D. N.D. N.D N.D. N.D. + hemagglutination no hemagglutination . = not determined :2 I + 0 u u 35 0m vocweumuov no: u .0.2 .Houucou muwowwwoomm Bduom m mm wow: 0-00 :Hmuum % .0.2 0N .0.2 00 m. 00 w oq £0-00 mucOSHMCH m w .a.z .o.z n w omma m. .a.z maza 0-0-mm m W 0s 0m 0m 00mm M 00mm M xn>02 n W 00 0N m w. 00~0 M ow~0.M 00s>02 saxoano “Hanna as->mH aunt HS->mH auan mo->nz aunt x->az aaaa amauos Hmauoc uwnnmu coxuwso coxowno ufinnmu moufi> muomwucm mo woufiu Hm .mumm mucooawcw 0cm .moumHomw >02 .00u>02 umcwmww mwomfiucm moonw> mo muouwu cowuwnwscw cowumcHuDwamaom .m OHan Immunodiffusion A single line of identity was formed between NDV-X, LNAF, BS-C-l LNAF, and NDV-GB, using chicken anti NDV-GB serum. (Fig. 8). Fig. 8. Immunodiffusion reactions. 1. 2 3. 4. 5 6 7. NAF . NDV-X NDV-GB LNAF BS-C-l LNAF IBV-42 Chicken anti-NDV-GB serum 37 38 Thermal stability 2; viral hemagglutinins Hemagglutinins present in allantoic fluids of NDV-GB, NDV-X, and BS-C-l LNAF, when tested with chicken erythrocytes, were not destroyed by heating at 56 C. (Table 4). Table 4. Thermal stability of viral hemagglutinins. HA titer after time (minutes) at 56 C Virus 0 30 60 90 120 NDV-GB 1280 2560 1280 640 640 NDV-X 1280 1280 640 640 640 88-0-1 LNAF 1280 1280 1280 640 640 Ether sensitivitngf NDV-X After treatment of NDV-X for 18 hours at 4 C with diethyl ether, its infectivity for chicken embryos was reduced at least 99.999%. (Table 5). Table 5. Ether sensitivity of NDV-X. Inoculum ELD50 per m1 é 4.0 NDV-X + diethyl ether 10 NDV-X control 109’2 39 25 stability 2; NDV-X Strain X of NDV was stable of pH 3.0 and labile at pH 11.0. (Table 6). Table 6. pH stability of NDV-X. Virus pH TCIDSO per m1 NDV-x + glycine-HCl 3.0 105-5 . 1.0 NDV-X + g1yc1ne-NaOH 11.0 10 NDV-X control (HBSS) 7,0 106'0 Sensitivity of 88-0-1 cells for the detection 2; NE! The BS-C-l cell line was extremely sensitive in detecting NDV. Inputs of as few as 101 ELDSO NDV-X, based on serial dilution, pro- duced CPE within 48- to 72 hours after inoculation. Similar results were obtained when 101 ELDSO NDV-X were combined with 106 ELDso of strain 42. However, no CPE was observed in uninoculated cultures, or in cultures inoculated with 106 ELDSO of strain 42 alone. The CPE could be prevented by reacting the viruses with NDV-GB chicken antiserum. Growth of NDV4X in chicken embryo kidney cell cultures Plaques, approximately 2 mm in diameter, were produced by NDV-X on chicken embryo kidney cell monolayers after incubation for 5 days. The titer was approximately 1.67 x 108 PFU per ml. (Fig. 9). Fig. 9. Plaques produced on CEKC by NDV-X 5 days after infection. 40 41 Viral interference Based on results of serum neutralization and hemagglutination inhibition tests, an excess of IBV interfered with the replication of NDV-X in chicken embryos. (Table 7). ms vocHEuouov uoa u .0.2 Eamon coxofiso .0.2 .0.2 on N.0 W 5.0 00u>02 fluam + Nu>02 000 + >0H 000 afiwmm coxowno an» on an» o.m m.a as->mH anew + xu>02 000 + >0H 000 .0.2 .0.2 on N.0 w N.m xu>02 000 + >0H 000 Bauom Eouom coxowno coxowno Eamon coxowno abu6m coxowno >02 Huam >0H Hucm >02 wuss >0H Hucm . nuwz wouw0=ooaw mo>unao Eoum Hm <2 moowwafi coaumNfiamuuooz woumo>umn "mauw> .mozunao aoxowno a“ xu>0z mo coHuMOflHQOw mnu co >0H >0 oudoummuouafi wcwumuumcoaov mumou coHananH ooHumcHuDwamamn 06m coauMNMHOHuSOG Eouom .m OHQmH DISCUSSION Infectious bronchitis virus (IBV) strains 41, 42, and 46 are apparently incapable of replication within serially propagated mammal- ian cells. During efforts to induce multiplication of IBV in a non- avian system, several types of cells, including cancerous, normal, epithelial, fibroblastic, adult, embryonic, diploid, and heteroploid were used, but there was no evidence of replication of the virus. Some animal viruses are capable of infecting a wide range of hosts, whereas the host Spectrum of other viruses is quite restricted. Infectious bronchitis virus, for example, commonly infects only the chicken. Newcastle disease virus (NDV), on the other hand, is capable of infecting many species, including chickens, turkeys,guinea fowls, ducks, geese, parrots, pheasants, sparrows, crows, martins, and several others, including man. Recent evidence of Newcastle disease virus infection in several previously unreported Species indicates that the host Spectrum of NDV is expanding, perhaps in part due to adaptative mutation (12). In short, NDV is an evolving pathogen (29). A prerequisite for virus infection of any cell is the attach- ment of that virus to the cell by receptors present on either the virus particle itself or the host cell. In general, the attachment procedure for animal viruses proceeds in two stages, the first of which is a rather loose, reversible stage, while the second is a firm, irreversible binding. One method of determining whether a virus has adsorbed to a cell is to measure the amount of free virus present in 43 44 the inoculum at different times following the addition of a known number of viral particles. If the amount of cell-free virus decreases over a period of time, it may be assumed that a certain percentage of the virus has attached to the cells. Another method used to determine whether virus has attached to cells is through electron microsc0py. Attachment of the virus to the cell, however, is not the only requirement for viral infection. The virus must then penetrate the cell, either by a passive process, or by actively weakening the cell membrane enzymatically in order to facilitate entry. However, once inside the cell, the virus may not be broken down into its subunits, or if it is broken down, its nucleic acid may not possess the necessary information to instruct the cell to begin production of new viral particles. On the other hand, the viral genome may supply only enough information to construct partial, or incomplete virus, resulting largely in abortive infections. Therefore, in the case of IBV and its interaction with mammalian cells, it would seem that three possibilities exist: First, there could be incompatibility of receptor sites on IBV and/or the mammalian cell, thus preventing attachment altogether; second, IBV could attach to, but not penetrate the cell, or; third, the virus could attach to and penetrate the cell, but would either fail to replicate or would undergo abortive infection. Because IBV commonly infects only the chicken, it would seem that it would possess receptors only for avian cells, and therefore would be unable to attach to cells of non-avian origin. Certain experiments were conducted with IBV and BS-C-l cells 45 in the presence of a "high magnesium" maintenance medium. It was assumed that by varying the ionic concentration of the medium, and thus perhaps altering electrostatic charges on either the virus or the cell, that attachment of the virus to the cell could be faciliated. Experiments of this nature were conducted by wallis and Melnick (60) using poliovirus. It was first believed by these investigators that a medium with a high concentration of divalent cations increased the rate of adsorption of poliovirus to the cells, but subsequent studies indicated that there was no change in the rate of adsorption. Instead, it was found that infective virus was released at a more rapid rate from cells exposed to high concentrations of magnesium ion in the maintenance medium. A similar effect was observed in the present study with NDV-X in BS-C-l cells. Infective virus was obtained more rapidly from cells exposed to high levels of magnesium ion than from cells exposed to a normal ionic complement. Although efforts to induce replication of IBV in mammalian cells were unsuccessful, a finding of significant importance arose from these studies. A virus, subsequently identified as Newcastle disease virus, whose characteristics very closely resembled those of the GB Strain, was isolated from certain allantoic fluid stocks of strain 42 and from a sample of apparently "normal allantoic fluid." The problem of latent viruses in chicken embryos, animals, and tissue cultures is of great importance to the virologist. Simian virus 40, for example, was not discovered until Rhesus monkey kidney cells were cultured in yitrg. Because the animal virologist cannot work with a chemically defined cultural medium, the presence of latent biologic 46 agents may adversely affect experimental results with viruses under intentional study. Workers who employ chicken embryos in their research may in- advertantly encounter one of the egg-borne diseases of birds. Endogenous bacterial diseases may be expected to produce deleterious results that would be obvious. Latent viral agents, however, are ‘“mmsur more insidious and perhaps may be transferred in serial passage with the known virus without being apparent to the investigator. Similarly, vaccines prepared from chicken embryos could become contaminated with these endogenous viral agents and therefore escape detection (14, 19, 34, 65). It may be possible that certain latent animal viruses could exist in a lysogenic state, in that the viral genome may be trans- ferred as "provirus" from generation to generation, as in the case of certain bacteriOphages. The circumstances which would initiate the change from a lysogenic to an active state are unknown, but might be brought about by introducing the provirus into another susceptible host, or by otherwise altering its natural environment. It is also feasible that, in the presence of certain inhibitors, the synthesis of materials for the virus under study may be prevented, thus allowing the latent virus to express itself (48). It is obvious that the conditions which contribute to the latency of infectious agents will require extensive investigation into the mechanisms of this biological phenomenon. Once the reasons for latency are understood, and the ensuing problems are solved, it may be possible to eliminate the word "latent" from the biologist's 47 vocabulary. The question of whether ovarian transmission of endogenous biological agents occurs has never been settled to the complete satisfaction of many investigators, since the evidence for such transmission is largely circumstantial. It would seem likely, how- ever, that if such a form of contamination did occur, it would take place in two possible ways: Either during egg formation or by in- vasion of the egg by the contaminant after laying. The most likely form of contamination by the latter method would be by contact with fecal material, whereas several possibilities exist for contamination of the egg during its formation. These would include infected germ cells, maternal blood, contaminated yolk, peritoneal fluid and cells, cells and secretions of the oviduct, infected semen and male feces, intestional tract contents, and infection from vents of picking birds (19). Evidence implicating ovarian transmission of viral diseases may come from various sources. Flock isolation studies, for example, indicated that avian lymphomatosis developed in flocks started from hatching eggs introduced into new and isolated poultry quarters (61). Such a finding would suggest that the virus may already have been present in the eggs prior to hatching. Incubator exposure studies and experiments in family isolation could also be used to help substantiate the possibility of ovarian transmission of certain avian diseases (61, 62). On certain occasions it may be impossible to detect the presence of a disease within a given flock since the birds may be asymptomatic 48 carriers. Newcastle disease, for instance, is usually detected by the presence of certain Specific clinical symptoms or by a variety of laboratory tests, including clinical examination, isolation of the virus from symptomatic birds, serologic examinations, and specific immunity tests (12). If the typical central nervous system manifestations of Newcastle disease are lacking, it is often difficult to distinguish ND from other diseases such as infectious bronchitis, laryngotracheitis, coryza, mycosis, leukosis, and pullorum disease (12). On certain occasions, however, there may be a complete absence of any disease symptoms within a flock. A situation of this nature occurred recently in Ireland (46). Random samplings of sera from the flock, however, contained antibody specific for NDV. The virus was eventually isolated from the feces of the asymptomatic birds. Because NDV-X was isolated from a supposedly normal sample of allantoic fluid it is possible that an asymptomatic, or sub-clinical, NDV infection existed in the commercial flock from which hatching eggs are obtained for research at Michigan State University. Vaccines against the common avian viral diseases are not used, and the flock is pullorum-tested only once a year. However, there has been no clinical evidence of any disease in this flock for several years. Egg production and hatchability have been normal (53). The laying flock, which is closed, is kept for only one year, after which it is sold to a commercial food processing company. In order to reduce the risk of introducing disease into the remaining flock the poultry handlers of the commercial company, who may have been in contact with diseased birds, are not 49 permitted near the remaining flock. Instead, the birds are delivered to the buyer outside the immediate premises. No birds, which may be infected, are imported, and no custom hatching for outside concerns is permitted. Although no overt symptoms of ND existed in this commercial flock, it is interesting to note that at approximately the same time the sample of "lethal normal allantoic fluid" was harvested, in November of 1966, difficulties were experienced in preparing CEKC monolayer cultures. Several of the embryos were dead at the 17th day of incubation, and many of the kidney cells from the living embryos did not attach to the petri dish surface. Although direct proof is lacking, it seems possible that a correlation exists between the isolation of NDV from normal allantoic fluid and the nearly simultaneous difficulties encountered with the chicken embryo kidney cell cultures. This would suggest quite strongly that a sub-clinical NDV infection may have existed in the flock at this time. The possible origin of this in- apparent infection is unknown, but the disease may have been introduced into the flock by wild birds which, according to other investigators, may act as carriers of Newcastle disease (26, 30, 43, 45). A sample of strain 42, dated March 1, 1961, was found to contain NDV. Because of illness in the family, the hatchery from which chicken embryds were obtained at that time had long since gone out of business, so that records concerning the health of the flock are not available. However, there were no reports of disease conditions in the flock, and no proof, therefore, can be submitted that ND, either clinical or sub- clinical, existed. 50 The flock, from which the hatching eggs were obtained, however, may have been exposed to NDV, since other varieties of poultry were maintained on the premises, and some of these other birds may have harbored a latent ND and transmitted the infection to the laying flock. It is, of course, possible that a chance laboratory contamination may have occurred while working with cultures of IBV and NDV-GB in this laboratory. This is extremely unlikely, however, Since utmost pre- cautions are taken so as not to employ IBV and NDV in experiments at the same time. Interference by IBV on the replication of NDV reported by other investigators (7, 9, 27, 28, 49) has been reconfirmed in this study. This interference takes place only in an avian system in which both IBV and NDV can replicate. Suppression of NDV by IBV may help to explain the reasons for its existence as a latent virus in cultures of IBV strain 42. The usual methods for detecting NDV in allantoic fluid are serum neutralization, hemagglutination, and hemagglutination in- hibition tests. Because NDV—X was probably present in very low levels in both the sample of strain 42 and LNAF, it would be impossible to detect its presence by hemagglutination, since it requires a minimum of approximately 106 NDV particles (12) to induce hemagglutination of chicken erythrocytes. Serum neutralization tests would be of no benefit in detecting NDV in a sample of IBV, since interference of NDV by IBV in an avian system would suppress its multiplication. Laboratory methods for the detection of NDV are adequate pro- viding sufficient numbers of viral particles are present to induce hemagglutination. If these conditions are not met, the virus might not 51 be recognized. Failure to detect NDV as a contaminant is avian vaccines poses a threat to an immunization program, since NDV could inadvertantly be introduced into a flock. In order to detect low levels of NDV in either a commercial IBV vaccine or a supposedly pure sample of virus by tissue culture methods it is necessary to employ cells which will support the replication of NDV and prevent multiplication of IBV. The choice of which cell lines to employ is important, not only in detecting NDV in a sample of IBV, but for the detection of other contaminating viruses in other systems as well. Poliovirus, for example, will not replicate in most rodent cell lines, and the use of such cells in detecting live poliovirus contaminants in certain vaccines, therefore, would be pointless (4). Based on the sensitivity of 38-0-1 cells to low levels of NDV, it is suggested that commercial IBV vaccine preparations be screened in these cells for the possible presence of NDV as an added step in vaccine quality control. The expense and additional time required to utilize this procedure are minimal considering that the loss of a pro- ductive flock through the introduction of latent disease is of much greater economic importance. The mechanism of interference between IBV and NDV, like other interference phenomena between other viruses, is not fully understood. Based on experimental findings of other investigators it appears that IBV interferes with NDV intracellularly. It may be possible, for ex- ample, that the information contained in the IBV genome may code for the production of an inhibitor which could prevent the uncoating or degredation of a superinfecting NDV particle. It is also possible 52 that certain nucleases could be produced by the IBV-infected cell which would destroy the nucleic acid moiety of the NDV particle, thus render- ing it incapable of replication. It is apparent that, as further infor- mation regarding the intracellular fate of viruses is made known, several questions concerning the mechanisms of interference will be answered. SUMMARY Infectious bronchitis virus strains 41, 42, and 46, do not replicate in FL, AV-3, L-929, H.Ep.#2,.KB, HEK, or BS-C-l mammalian cell lines, based on CPE and "blind passages." A virus, subsequently identified as NDV with characteristics Similar to the GB strain, was isolated from certain stocks of supposedly pure IBV strain 42, as well as from a sample of "normal allantoic fluid". Serum neutralization tests, hemagglutination inhibition, immunodiffus- ion, electron microscopy, and comparison of certain properties with those of known NDV were used to establish the identity of the virus isolates. Interference, exerted by IBV on NDV in a system in which both viruses can replicate, such as the chicken embryo, may have prevented earlier detection of the latent NDV contaminant. The BS-C-l cell line, although unable to support the replication of IBV, is extremely sensitive in detecting NDV, even when combined with a high excess of IBV. 53 10. ll. 12. LITERATURE CITED Ablashi, D. V. 1962. Chick-embryo-lethal-orphan (CELO) virus as a tissue culture contaminant. Thesis. University of Rhode Island, Kingston, Rhode Island. , P. W. Chang, and V.J. Yates. 1965. The effect of a latent CELO virus infection in the chicken embryo on the propagation of Newcastle disease and influenza viruses. Avian Dis. 2: 407-416. American Public Health Association. 1964. "Diagnostic Procedures for Viral and Rickettsial Diseases", 3rd ed. E. H. Lennette, ed. New York. American Type Culture Collection. 1967. "Registry of Animal Cell Lines". American Type Culture Collection Repository, Rockville, Maryland. 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