Structural and Growth Characteristics of Infectious Bursal

Transcription

1 JOURNAL OF VIROLoGY, Apr. 1976, p Copyright X 1976 American Society for Microbiology Vol. 18, No. 1 Printed in U.S.A. Structural and Growth Characteristics of Infectious Bursal Disease Virus H. NICK, D. CURSIEFEN, AND H. BECHT* Institut fur Virologie, Justus Liebig-Universitat, Giessen, Germany Received for publication 6 October 1975 The infectious bursal disease virus is not enveloped and has a diameter of 60 nm and a density of about 1.32 g/ml. It contains two pieces of single-stranded RNA with molecular weights close to 2 x 106. The capsid is made up of four major polypeptides with molecular weights of 110,000, 50,000, 35,000, and 25,000. The virus replicates in chicken embryo fibroblasts rather than in epitheloid cells. After an eclipse period of 4 h, virus production reaches a maximum about 12 h later. The virus has no structural or biological similarities with defined avian reoviruses, and it cannot be classified in one Qf the established taxonomic groups. There have been many contradictory reports about the structural and biological characteristics of the causative agent of infectious bursal disease (IBD) (Gumboro disease) of chickens and about the taxonomic position of the virus. It has been designated as a picornavirus (3) or an adenovirus (1), or more frequently it has been placed among the reovirus-orbivirus group (7-9, 17). Most of these studies have been confined to the morphological appearance ofthe virus particle. We recently described a virus, which had been incriminated as being an etiological agent of IBD (9), as a true reovirus, but it was not possible to attribute any definite pathological significance to it (15). Therefore, we pursued these studies in an attempt to define the etiological agent of infectious bursitis. The virus strains described in this communication can be regarded as causative agents of this disease with high certainty, but they do not have the structural and biological properties of reoviridiae. MATERIALS AND METHODS Viruses and cell cultures. IBD virus strain 1/PV was kindly provided by G. Mandelli, Milan, as passage 25 in embryonated eggs. Strain Cu-i was isolated in this laboratory from a pooled tissue homogenate of the Bursa of Fabricius (BF) from birds that had died from infectious bursitis or were killed after they had contracted the disease. Infectious material was inoculated perorally into a specific pathogen-free chicken at 3 weeks of age. When the animal died 3 days later, a tissue sample of its BF was homogenized and inoculated into chicken embryo fibroblasts (CEF), which had been prepared according to standard procedures. When a full cytopathic effect had developed, the medium was diluted serially and a plaque assay was carried out. An individual large plaque was picked after 2 days of incubation at 40 C, and the virus underwent five more plaque passages before it was propagated again in CEF and stored frozen at -70 C. Avian reovirus strains 2207/68 and Wi, the Dearing strain of reovirus type 3, and the methods applied for their growth and purification were the same as described in a previous communication (15). The DEK strain of pseudorabies virus had been 227 grown in rabbit kidney cells as described (13). The Rostock strain of fowl plague virus was grown in embryonated eggs. Purification of virus from BF and CEF. Infected bursae were collected from birds that had been inoculated at 3 to 5 weeks of age either with tissue homogenates from field cases or the isolated Cu-i strain. The animals were usually killed about 48 h postinfection, and the mostly enlarged, hemorrhagic and edematous organs were homogenized as roughly a 20% suspension in Tris-borate buffer (0.1 M boric acid, M Tris, 0.1 M NaCl, ph 8.2) and treated with the fluorocarbon Frigen 113 (Farbwerke Hoechst) at a ratio of about 5:1 for 5 min in an ice bath. After centrifugation at 3,000 rpm for 20 min at 4 C, any residual fluorocarbon was removed by shaking and phase separation with ether. The virus was centrifuged from the aqueous phase by ultracentrifugation at 22,000 x g for 3 h. CEF, infected at a multiplicity of about 1, were scraped from the bottom of Roux flasks or roller bottles into the medium when a cytopathic effect was clearly visible. The cells were homogenized, treated with fluorocarbon, and centrifuged as described above. The virus pellets from either source were resuspended in SSC buffer (0.015 M sodium citrate, 0.15 M NaCl, ph 7.2) and centrifuged through a 10 to 60% sucrose gradient (wt/wt, in 1 x SSC) in an SW27 rotor at 25,000 rpm for 3 h at 4 C or in an SW41 rotor at 35,000 rpm for 2 h at 5 C. Fractions of 1 ml were collected from the bottom of the tube and assayed for infectivity by plaque assay

2 228 NICK, CURSIEFEN, AND BECHT in CEF. The virus-containing fractions were centrifuged again, and the virus pellet was resuspended in buffer and mixed with CsCl to give a density of 1.32 g/ml. The mixture was centrifuged in an SW65 rotor at 50,000 rpm for 22 h at 15 C. Fractions of 0.5 ml were either collected from the bottom or the visible bands were drawn through the sides of the tubes. Labeling the virus nucleic acid. Roller bottles containing monolayers of CEF were infected with 1/ PV virus at a multiplicity of approximately 1. After 1 h at 37 C, the inoculum was removed, and incubation was continued with Eagle medium without serum. Ninety minutes later, 1 mci of [3H]uridine or [3H]thymidine was added. When a cytopathic effect appeared about 24 to 36 h later, cells plus medium were harvested and the virus was purified by treatment with fluorocarbon and sucrose density gradient centrifugation. Fractions of 1 ml were assayed for infectivity and radioactivity. RNase sensitivity of viral RNA. Labeled RNA was extracted from the purified virus preparation with sodium dodecyl sulfate and phenol. The ethanol precipitate was dissolved in 2x SSC and divided into two aliquots. One aliquot received 10,g of pancreatic RNase per ml and was incubated at room temperature for 20 min. Labeled complementary strands of influenza virus RNA (20) were included as RNase-sensitive controls. Radioactivity precipitable by 6% trichloroacetic acid was determined in RNase-treated and in control preparations by liquid scintillation. Analysis of viral RNA in polyacrylamide gels. Polyacrylamide gels (7.5%), which contained 6 M urea, were prepared according to Martin and Zweerink (14) and were run under the conditions described for the analysis of reovirus RNA (15). Virus samples were dissociated in 2 M urea, 1% sodium dodecyl sulfate, and 0.1% mercaptoethanol at 55 C for 60 min and applied directly to the gels. A purified preparation of reovirus type 3 was used as marker. In additional assays 2.5% polyacrylamide gels were used, which contained 0.5% agarose and no urea. The buffer system had been described by Loening (12). Tobacco mosaic virus RNA and plant 25s, 23s, 20s, 18s, and 16s rrna (10, 11) were used as markers. All gels were stained with methylene blue (16). Polypeptide composition of virus particles. The protein composition of the virus was determined in 7.5% polyacrylamide gels as outlined by Smith et al. (21). The gels contained 5 M urea; the buffer was 0.1 M sodium phosphate, ph 7.2. The virus samples were dissociated with 6 M urea, 1% sodium dodecyl sulfate, and 0.1% mercaptoethanol in a boiling-water bath for 2 min. Electrophoresis was carried out at 6 ma/gel for 20 h. Serological analysis. Birds that had survived after being infected with bursal homogenates from field cases or purified Cu-i virus were bled 2 weeks postinfection, and their serum was stored at -20 C. Rabbit hyperimmune serum was prepared by subcutaneous inoculation of a purified virus preparation J. VIROL. from BF tissue, which had been incorporated into Freund complete adjuvant. A second injection was applied 3 weeks after the first one, and blood was drawn 2 weeks later. Antigens for gel diffusion were either homogenized BF from infected birds or extracts from infected CEF prepared by freezing and thawing of cells that had just shown a cytopathic effect. Neutralization tests were carried out by mixing approximately 100 PFU with serially diluted antiserum and inoculating the mixture after 30 min at room temperature into CEF. Plaques were developed by staining with neutral red after 2 days. The highest serum dilution that reduced the plaque count by more than 80% was taken as the end point. For staining infected CEF with fluorescent antibodies, the gamma globulin fraction of a chicken convalescent serum (neutralization titer, 1:16,000) was conjugated with fluorescein isothiocyanate. Nonconfluent monolayers grown on cover slips were fixed with acetone and stained as described (2). Reagents. 5'-Bromodeoxyuridine was purchased from Serva, cytosine arabinoside was from Nutritional Biochemicals, and bovine pancreatic RNase was from Sigma. RESULTS Pathogenicity and serological properties of viruses. The virus strain Cu-i had undergone several plaque passages in CEF at limiting dilutions and consisted of a homogeneous population of virus particles in electron micrographs. Inoculation of the plaque-purified strain into susceptible chickens caused an overt and rapidly progressing disease, which was mostly fatal and showed the pathognostic lesions of Gumboro disease in the BF. Serum samples from surviving individuals produced precipitates in agar gels with bursal extracts from field cases or artificially infected birds or with cell extracts from infected CEF. Lines of identity also formed when a rabbit serum, produced by immunization with a bursa-derived field virus preparation, diffused against these antigens. Convalescent sera from chickens infected with strain Cu-1 or 1/PV cross-neutralized either strain completely (Table 1). The table also shows that the rabbit immune serum neutralized both strains at identically high titers. Control sera from normal specific pathogen-free animals did not neutralize any infectivity. Rabbit and chicken sera directed against the avian reovirus 2207/68 (15) neutralized neither one of the two IBD virus strains nor did it produce any precipitates in agar gels when it diffused against the IBD virus-specific antigens. All these experimental data show that the virus strains used were etiological agents of IBD. Growth of virus in CEF. Reports (6, 18, 19) that IBD virus replicates in CEF with production of a cytopathic effect could be confirmed for strains Cu-1 and 1/PV. The growth curve of Fig. 1 shows that after an eclipse period of about 4 h virus titers increased to reach a maximum of 107 to 108 PFU/ml at 10 to 16 h postin-

3 VOL. 18, 1976 TABLE 1. Seraa Neutralization assays ofibd virus strains Cu-i and 1/PV Neutralizationr of virus strain: Reovirus Cu-i 1/PV 2207/68 Chicken anti-cu-1 8,000 8,000 0 Rabbit anti-cu-1 32,000 32,000 0 Chicken anti-reovirus 0 0 1, /68 Rabbit anti-reovirus /68 Chicken normal se rum a Chicken anti-cu-1 serum, serum sample collected from a surviving bird 14 days postinfection. Rabbit anti-cu-i serum, prepared by hyperimmunization with virus purified from the BF of chickens infected with strain Cu-1. The sera against avian reovirus 2207/68 was prepared in chickens and rabbits as described (15). Normal chicken serum was pooled from specific pathogen-free animals. b Neutralization is expressed as the reciprocal of the highest serum dilution reducing plaque counts by more than 80%. E07 l l Hours After Infection FIG. 1. Growth curve of IBD virus strain Cu-i. CEF were inoculated with undiluted culture fluid of CEF after a clear cytopathic effect had developed. After an adsorption period of 1 h individual plates were withdrawn at hourly intervals and assayed for infectivity in medium and in cells after freezing and thawing. Plaque titrations were done in CEF. Symbols: *, extracellular virus in medium; *, cell-associated virus. section. Infection of cells derived from the chorioallantoic membrane of embryonated eggs (4) yielded less virus than a conventional culture of CEF. Examination of infected cells with fluorescent antibodies confirmed that IBD virus replicated in typical fibroblasts rather than in cells of the epitheloid type. Punctiform fluorescence usually appeared in fibroblasts around 6 INFECTIOUS BURSAL DISEASE VIRUS 229 h postinfection in perinuclear areas and became patchlike and highly intense with a diffuse background in the whole cytoplasm as the infection progressed. Purification of virus and density of virus particles. Virus could be purified from the BF of chickens infected with field material, strain Cu-1, or strain 1/PV after treatment of the tissue homogenates with fluorocarbon and phase separation with ether. Infectivity was not destroyed by this treatment, which is in accordance with many observations (9, 18). When the virus that had been concentrated from the aqueous phase was run through a sucrose gradient, two distinct bands were usually visible in the central region of the tube, which corresponded to 40% sucrose (Fig. 2). When the bands in the sucrose gradient were collected separately by puncturing the tubes from the FIG. 2. Sucrose density centrifugation of IBD virus, which was extracted from the BF of birds infected with field material and concentrated by ultracentrifugation. The material was loaded onto a 10 to 60% sucrose gradient and centrifuged in an SW27 rotor at 25,000 rpm for 3 h at 4 C.

4 230 NICK, CURSIEFEN, AND BECHT side and the virus was centrifuged into equilibrium in CsCl, a density of 1.32 to 1.33 g/ml was determined for each band from the sucrose gradient (Fig. 3). A second band with a density of 1.30 g/ml was regularly visible in CsCl gradients. This fraction contained mostly particles in various stages of disintegration when it was examined in the electron microscope. Sometimes another band was visible with a density of g/ml, containing morphologically indistinct debris. This behavior in sucrose and CsCl gradients was identical for virus purified from BF or from CEF. Since the two bands in the sucrose gradient had identical densities, they were routinely pooled for further purification in CsCl. 0) HE. -6- a) cj E I-I v) a) Fraction number FIG. 3. Equilibrium density centrifugation ofibd virus strain Cu-1 grown in CEF in CsCl. The two visible bands of sucrose gradients were pelleted, resuspended in buffer, mixed with CsCl to give a density of1.32 g/ml, and centrifuged in an SW65 rotor at 50,000 rpm for 22 h at 15 C. Fractions of 0.5 ml were tested for infectivity by plaque assay in CEF, and their density was determined by weighing a calibrated volume of 25 p1. Symbols: 0, density (grams per milliliter); *, infectivity (PFU per milliliter). Morphology of the virus. Electron micrographs (Fig. 4) of a virus preparation purified from the BF by sucrose density centrifugation and stained with phosphotungstic acid showed non-enveloped particles of icosahedral symmetry with an average diameter of about 60 nm. An outer shell could not be seen. Many particles had the appearance of empty shells or were in various stages of disintegration. Some of the capsomeres displayed the form of hollow cylinders. Type of nucleic acid. In the presence of 50 or 100 ug of 5'-bromodeoxyuridine or 10 tug of cytosine arabinoside per ml, growth of IBD virus proceeded normally in CEF, whereas the replication of pseudorabies virus was totally suppressed under these conditions (Table 2). Figure 5 lends further proof to the assumption derived from these experiments that the viral genome is RNA. It can be seen that the highest number of [3H]uridine counts coincides with maximal infectivity in a sucrose gradient, whereas the [3H]thymidine activity of a parallel tube in the same run stayed on background levels in the vicinity of the infectivity peak. The [3H]uridine-labeled RNA extracted from' a purified preparation was rendered acid soluble by RNase (Table 3). It was not degraded to quite the same extent as a labeled' influenza virus RNA, which had been included as a control, but an avian reovirus RNA largely resisted the 10-fold concentration of the same enzyme under identical experimental conditions (15). One can therefore assume that IBD virus RNA is single stranded, perhaps with some backfolded regions. In an attempt to J. VIROL. determine the molecular weight of virus RNA, purified virus preparations were analyzed in polyacrylamide gels un- TABLE 2. Growth ofibd virus in the presence of DNA inhibitors PFU/ml Virus strain No in- Ara-C No in- BUdR hibitor hibitor Bd 1/PV 1 x x x x 107 Cu-i 1 X X X X 107 FPV 2 x x 107 NT NT PsR 3 x X X x 10' a Culture media of CEF contained 10 ug of cytosine arabinoside (ara-c) or 100 pg of 5'-bromodeoxyuridine (BUdR) per ml during the whole replication cycle. Infectivity of media was tested by plaque assays at 24 h postinfection for Cu-i and 1/PV, at 8 h for fowl plague virus (FPV), and at 24 h for pseudorabies (PsR) virus. The effect of the two inhibitors was tested in different culture preparations of CEF. NT, Not tested.

5 VOL. 18, 1976 INFECTIOUS BURSAL DISEASE VIRUS 231 f~ ~ ~ ~ ~ 7 w FIG. 4. Electron micrograph of IBD virus. Magnification, x150,000. The virus had been extracted from the BF of birds infected with field material and purified by sucrose density centrifugation. -D LL 0l- Fraction Number FIG. 5. Incorporation of [3H]uridine into the infectious fractions of a sucrose gradient. CEF infected with IBD virus were pulsed with [3H]uridine or [3H]thymidine. Virus from culture medium and cells was pelleted, and the resuspended material was centrifuged through a sucrose density gradient in an SW41 rotor. Fractions of 1 ml were assayed for radioactivity and infectivity. Only plaque numbers of the tube containing [3H]uridine are shown; infectivity of the [3H]thymidine-labeled sample was virtually identical. Symbols: *, [3H]uridine; A, [3H]thymidine; *, infectivity (PFU per milliliter in CEF). der different conditions. Figure 6 shows that in a 7.5% gel two clearly distinct bands could be stained with methylene blue near the top of the gel. The methods were those employed for the analysis of reovirus RNA, and it is clear by comparing the 10 segments of the double- E CL TABLE 3. RNase sensitivity of viral RNAa Source of RNA RNase present 3H dpm IBD virus - 4,370 IBD virus FPV - 25,360 FPV a Phenol-extracted RNA of 1/PV grown in CEF and labeled with [3H]uridine was treated with 10 jig of pancreatic RNase per ml at room temperature for 20 min. Fowl plague virus (FPV) RNA was added as a single-stranded control. Radioactivity precipitated by trichloroacetic acid was counted by liquid scintillation. stranded RNA of a true reovirus that the structure of IBD virus RNA is totally different from reovirus RNA. Also, in a 2.5% polyacrylamideagarose gel two bands that migrated slightly faster than tobacco mosaic virus RNA could be resolved. Based on the markers that comigrated in Fig. 7, the molecular weights of the two RNA pieces of IBD virus RNA were determined as 1.8 x 106 and 1.75 x 106. There was no indication of a larger RNA in the upper regions of 2.5% acrylamide gels carrying IBD virus RNA. Polypeptide composition. Four polypeptides could generally be stained with Coomassie brilliant blue in 7.5% polyacrylamide gels in which CEF-grown virus or virus purified from the BF of chickens infected with field material or

6 232 NICK, CURSIEFEN, AND BECHT FIG. 6. Acrylamide gel analysis ofibd virus and reovirus RNA. Purified virus was dissociated with sodium dodecyl sulfate and mercaptoethanol and applied directly to a 7.5% acrylamide gel containing 6 M urea. Electrophoresis was carried out for 24 h at 4 ma/tube. Gels were stained with methylene blue. (Left) IBD virus purified from the BF of birds infected with field material. (Right) Avian reovirus strain Wi. strain Cu-1 was analyzed. A representative example is shown in Fig. 8. The faintly and irregularly stained region just below VP-1 probably represents an artifact, because it was absent in all the other gels. The molecular weights of the four structural components corresponded to 110,000 (VP-1), 50,000 (VP-2), 35,000 (VP-3), and 25,000 (VP-4). They are based on the migration distances of the known structural components of reovirus type 3 (21) and the positions of serum albumin, ovalbumin, and myoglobin in identical gels. Sometimes the minor component VP-2 appeared as a double band, and other preparations produced a band (VP-0) in a posi- tion corresponding to a molecular weight of approximately 180,000. Figure 9 shows an example in which the two types of variations showed up in the electrophoretic analysis of the same preparation of CEF-grown virus. It cannot be decided with certainty whether these two additional components represent minor structural entities of the virion or occasional contaminants. DISCUSSION The appearance of typical symptoms and lesions of infectious bursitis after inoculation of plaque-purified virus into susceptible birds and the production of specific antibodies in such animals justify the assumption that Cu-1 represents a pure strain of the etiological agent of this disease. It is obviously s'-rologically identical with strain 1/PV, which has been considered a causative agent of IBD (18, 19). Virus preparations could be obtained by purifying the virus from infected BF or by growing it in CEF. Electron micrographs of such preparations produced pictures essentially identical to those observed by others (1, 7, 8), in particular, the size of the icosahedral virion, the absence of a second capsid shell, and the high proportion of disintegrating particles. It should be added, however, that particles were present in many microscopic fields for which it was difficult to exclude the presence of an outer shell. Our electron micrographs did not permit us to draw any conclusions with respect to the triangulation of the IBD virion, because capsomeres with a five- or sixfold symmetry on one particle could not be delineated. A density of 1.32 to 1.33 g/ml, which has been 10o E 105 _ 0) D - TMV J. VIROL. 3 I b Relative Migration Distance(cm) FIG. 7. Determination of the molecular weight of IBD virus RNA by electrophoresis in 2.5% polyacrylamide gels containing 0.5% agarose. The virus was dissociated as for Fig. 6. Electrophoresis was carried out in Loening's buffer system (12) for 8 h at 4 ma/ gel. Markers were tobacco mosaic virus (TMV) RNA and plant rrna. Gels were stained with methylene blue.

7 VOL. 18, 1976 VP I VP 2- VP 3 n- VP 4 FIG. 8. Polypeptide composition ofibd virus. Virus was purified from the BF of birds infected with field material by density gradient centrifugation in sucrose and CsCl. The 7.5% polyacrylamide gel contained 5 M urea; the buffer was 0.1 M sodium phosphate (21). Electrophoretic runs were at 6 ma/gel for 20 h. The gel was stained with Coomassie brilliant blue. measured for the infectious virus particle in all our preparations, is lower than 1.34 g/ml reported by Hirai and Shimakura (8). A density of approximately 1.32 g/ml corresponded to the visible band in CsCl gradients, which contained complete virus particles, coincided with maximal infectivity, and corresponded to the peak values of [3H]uridine incorporated into the infectious fraction. All our data, including the effect of DNA inhibitors on virus growth and incorporation of [3H]uridine into the infectious fractions of a gradient with concomitant exclusion of [3H]thymidine, indicate that the virus genome is RNA. The sensitivity towards RNase suggests that it is mostly single stranded. Analysis of the RNA in polyacrylamide gels shows that INFECTIOUS BURSAL DISEASE VIRUS 233 the genome consists of two pieces with almost identical molecular weights of approximately 2 x 106. A longer molecule could never be demonstrated, even in gels with the large pore size of 2.5% acrylamide. The two RNA pieces always appeared in identical positions in many repeated runs with different preparations of different origin. If, therefore, the two pieces represent artifacts of preparation, which cannot be excluded at present, fragmentation of a large RNA must occur at an identical weak spot of the RNA strand. It has not been determined yet whether the larger RNA segment is preferentially associated with particles of the band sedimenting faster in sucrose gradients and what the proportions are between the number of physical particles, possibly including defective interfering particles, and infectious units in the two isolated bands of the gradient. In view of the limited coding capacity of either RNA piece it appears probable that both are needed to form an infectious virion. It cannot be ruled out that a minority of particles, which represent the infectious virus, contains a larger RNA molecule that is below the level of delectability. In analogy to IBD virus and reovirus RNA, the polypeptide pattern of the capsids of IBD virus has no similarity with the protein composition of avian or mammalian reoviruses (15, 21) or orbiviruses (14). The experimental data available now justify the assumption that the virion of IBD virus is composed of at least four polypeptides. Further experiments and different conditions of gel electrophoresis have to FIG. 9. Absorbance tracings of a 7.5% polyacrylamide gel in which CEF-grown IBD virus strain 1/ PV had been analyzed under the conditions in Fig. 8. The gel shows an additional band, VP-0, and a double band in the region of VP-2 after staining with Coomassie brilliant blue.

8 234 NICK, CURSIEFEN, AND BECHT clarify whether VP-2 can constantly be resolved into two components and whether VP-0 may represent an uncleaved precursor protein that is erratically encapsidated. The growth characteristics of IBD virus and its host cell range in tissue culture are clearly different from avian reovirus strains 2207/68 and Wi, which require epitheloid cells for their growth and do not affect fibroblasts (15). If one summarizes the information available now, one has to conclude that IBD virus has only the approximate size in common with orbiviruses (22) and there is no resemblance between the genomes or the peptide composition of IBD virus and any representative of reoviridiae as defined by Fraenkel-Conrat (5). Moreover, antigenic structure, growth characteristics, and pathogenicity distinguish IBD virus from avian reoviruses. All this means that it is hardly possible to classify IBD virus with one of the established taxonomic groups. ACKNOWLEDGMENTS We are obliged to E. Weiss for his help with the electron micrographs and to H. L. Sanger and C. Scholtissek for providing the marker and control RNA. This work was supported by the Sonderforschungsbereich 47 (Virologie). LITERATURE CITED 1. Almedia, J. D., and R. Morris Antigenicallyrelated viruses associated with infectious bursal disease. J. Gen. Virol. 20: Becht, H Untersuchung uber die Biosynthese and uber den serologischen Nachweis des Ribonucleoproteid-Antigens von Influenzaviren. fl. Die Anwendung der indirekten Hamagglutinationsreaktion fur den Nachweis des RNP-Antigens und von hullspezifischen Antigenen. Z. Med. Mikrobiol. Immunol. 156: Cho, Y., and S. A. Edgar Characterization of the infectious bursal agent. Poultry Sci. 48: Cursiefen, D., and H. Becht In vitro cultivation of cells from the chorioallantoic membrane of chick embryos. Med. Microbiol. Immunol. 161: Fraenkel-Conrat. H Descriptive catalogue of viruses, p. 35, 41. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology, vol. 1. Plenum Press, New York. 6. Gelenczei, E. F., and P. D. Lunger Isolation of a J. VIROL. reovirus from Bursa of Fabricius of chicken affected by Infectious Bursal Disease. J. Am. Vet. Med. Assoc. 156: Harkness, J. W., D. J. Alexander, M. Pattison, and A. C. Scott Infectious bursal disease agent: morphology by negative stain electron microscopy. Arch. Virol. 48: Hirai, K., and S. Shimakura Structure of infectious bursal disease virus. J. Virol. 14: Kosters, J., H. Becht, and R. Rudolph Properties of the infectious bursal agent of chicken (IBA). Med. Microbiol. Immunol. 157: Loening, U. E Molecular weights of ribosomal RNA in relation to evolution. J. Mol. Biol. 38: Loening, U. E RNA structure and metabolism. Annu. Rev. Plant Physiol. 19: Loening, U. E The determination of the molecular weight of RNA by polyacrylamide-gel-electrophoresis. Biochem. J. 113: Ludwig, H., H. Becht, and R. Rott Inhibition of herpesvirus-induced cell fusion by concanavalin A, antisera, and 2-deoxy-D-glucose. J. Virol. 14: Martin, S. A., and H. J. Zweerink Isolation and characterization of two types of blue tongue virus particles. Virology 50: Nick, H., D. Cursiefen, and H. Becht Structural and growth characteristics of two avian reoviruses. Arch. Virol. 48: Peacock, A. C., and C. W. Dingman Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry 6: Petek, M., and G. Mandelli Proprieta biologhiche di un reovirus isolato da un focolaio di malattia di Gumboro. Atti Soc. Ital. Sci. Vet. XXII: Rinaldi, A., G. Mandelli, G. Cervio, A. Valeri, D. Cessi, and S. Pascucci Untersuchungen uber die Atiologie der sogenannten Gumboro-Krankheit. I. Isolierung und Charakterisierung des Virus, Embryopassagen und kunstliche Ansteckung des Kukens, p In IVth Int. Kong. der W.V.P.A., Kongreftbericht. Belgrade. 19. Rinaldi, A., G. Mandelli, D. Cessi, G. Cervio, and A. Valeri Untersuchungen uber die Atiologie der sogenannten Gumboro-Krankheit. II. Pathogene Wirkung des Virus auf einige Laboratoriumstiere, p In IVth Int. Kong. der W.V.P.A., Kongre,8bericht. Belgrade. 20. Scholtissek, C., and R. Rott Hybridization studies with influenza virus RNA. Virology 39: Smith, R. E., H. J. Zweerink, and W. K. Joklik Polypeptide components of virions, top component, and cores of reovirus type 3. Virology 39: Verwoerd, D. W Diplornaviruses: a newly recognized group of double-stranded RNA viruses. Prog. Med. Virol. 12: