- Email: [email protected]
The structure of infectious bronchitis virus
VIROLOGY
23, 403407
(1964)
The
Structure
of
Infectious
Bronchitis
Virus
D. M. BERRY Glaxo Research,
Greenford,
Middlesex,
England
J. G. CRUICKSHANK Department
of Pathology,
H. I’. CHU School of Veterinary
Cambridge AND
University,
England
R. J. H. WELLS
Medicine, Cambridge University, Accepted April 6, 1964
England
The structure of infectious bronchitis virus of chickens has been examined by the negative staining technique in the electron microscope. The particles are between 800 and 1200 A in diameter and bear projections on their surfaces. The effects of ether, sodium dodecyl sulphate, trypsin, and neuraminidase on the morphology and on certain biological properties of the virus were also examined. INTRODUCTION
Studies on the virus particle of infectious bronchitis of chickens have been few. The virus passes Berkefeld filters, grades V, N, and W (Beach and Schalm, 1939), but no accurate measurements of particle size have been made by filtration technique. Electron microscope studies on unstained and on shadowed preparations reveal spherical and “tadpole shaped” particles between 60 and 120 rnp in diameter (Reagan et al, 1948; Reagan and Brueckner, 1952). Chu (1961, unpublished work) demonstrated spherical particles in shadowed preparations apparently containing an outer capsule. In ultrathin sections of virus-infected chorioallantoic membranes, Domermuth and Edwards (1957) found numerous intracytoplasmic particles with dense outer portions and less dense centres giving a “doughnut” like appearance. Their particles were between 178 and 200 rnp in diameter. This paper reports high resolution electron microscope studies on the virus employing the negative staining technique of Brenner and Horne (1959). After the first figures of the virus had been obtained, attempts were made to split the particle to reveal
visible internal components. Various agents were used, and the particles were examined morphologically after each treatment. The structure of the treated particles was contrasted with the structure of a member of the influenza group of myxoviruses similarly treated. Further, as treatment of the particles with various agents may alter the surface properties of the virus, attempts were made to relate such alterations to changes in the morphology of the particles. For example, Corbo and Cunningham (1959) described haemagglutination of chicken erythrocytes by trypsin-treated virus, although under normal conditions infectious bronchitis does not haemagglutinate. MATERIALS
AND
METHODS
Four virus strains were examined. Two were strains isolated in England, Al63 by H. I’. Chu, and H17 by J. S. Garside. The other two strains employed in this study were the Connecticut strain and the Beaudette strain. All were cultivated in embryonated eggs by allantoic inoculation, harvests from which contained about lo6 EIDgO virus per milliliter. These virus harvests were con403
centrated by ultrac&rifugation for 1 hour at 54,000 9, aud the resulting pellets mere Wsuspended in 1 ‘/;i ammoniunl acetate. &asured portions were mixed with equal portions of 2 % potassium phosphotungstate (adjusted to pH 7.0 with 1% KOH) and t#he mixtures were either sprayed (with a Vaponephrin nebuliser) or dropped onto carboncoated grids. The grids were examined in the Siemens Elmiskop microscope at instrumental magnifications of 40,000 and 80,000 times with double condenser illumination. RESULTS
The virus was &bed for sensitilyity to c>t’her by the technique of Andrcwes and Horstrnann (19-19), and was found t,o be cthcr sensitive. For nlorphological studies a suspension of the egg-grown virus was shaken with an equal volume of ether overnight at room temperature. The ether was blown off with nitrogen, and the preparation was prepared as described above for microscopy. Particles were still recognisable and some retained some of their spikes, though most were partly disrupted. The damaged coats split open rather than disintegrated. The contents of some particles were released, but no morphological pattern could be made out (Fig. 5).
The particles from all four strains studied closely resembled each other. 11oderate numbers of particles were seen lying singly or in small clumps. The particles Effect of Xodium Dodecyl Sulphate showed considerable pleomorphism although Sodium dodecyl sulphate was used to try most of them were approximately circular in to split the virus, as Laver (1963) reported outline with diameters between 800 and 1200 the effectiveness of this agent in breaking up A (Fig. 1). Most but not all of the particles influenza particles. had projections from their surfaces. These A drop of virus concentrate was placed on “spikes” were often seen over part of the sur- a grid and fixed in formaldehyde vapour for face only and were less densely packed than 15 minutes. A drop of either 0.1% or 0.01% those seen in influenza viruses. They varied sodium dodecyl sulphate was placed on the considerably in shape. Commonly they ap- grid for 2 minutes and then blotted off. A peared to be attached to the virus by a very drop of 2% phosphotungstate was then put narrow neck and to thicken towards their on the grid for 2 minutes, and the excess distal ends, sometimes forming a bulbous fluid was drained off. mass 90-110 A in diameter (Figs. 2 and 3). Solutions containing 0.01% of the deterThese pear-shaped masses could sometimes gent removed all the spikes, but otherwise be seen lying on the surface of particles, ap- had no effect on the integrity of the parparently in clusters. In other particles the ticles (Fig. 6). spikes were more rod-shaped, but many were Solutions containing 0.1% of the deterbent in the middle. Spontaneous disruption gent produced partial or complete disruption was sometimes seen. That release of the in- of all virus particles. The coats appeared to ternal component can take place without break up into smallish pieces, but no rolling much damage to the outer coat is seen in up into rosettes was seen. Fig. 4, which shows an empty viral coat still In view of the removal of the spikes by lined with spikes. A proportion of the par- 0.1% sodium dodecyl sulphate, the haemagticles had invaginations in their centres. glutinating activity of the treated material Some of these were due to pooling of phos- was investigated using fowl erythrocytes. So photungstate on the loose outer coat, but in haemagglutinating activity could be demonothers the invaginations were well defined strated in sodium dodecyl sulphate-treated and were lined with a conspicuous mem- particles. brane. It was not clear whether the ringEflect of Trypsin shaped particles were truly of that shape or Haemagglutination of chicken erythrowhether the appearance was due to infolding cytes by trypsin-treated virus has been reof the membrane. ferred to above, and the morphology of tryp-
STRUCTURE
OF INFECTIOTJS
BRONCHITIS
VIRUS
405
FIG. 1. Infectious bronchitis virus showing the whole particles with the surface projections. Magnification: X 130,000. FIG. 2. A single infectious bronchitis virus particle showing the bulbous appearance of the surface projections. Magnification: X 200,000. FIG. 3. A particle of fowl plague virus (influenza A) showing the close-set cylindrical surface projections. Magnification: X 200,000.
FIG. 3. Two virus particles, one showing an intact membrane to which the projections remain adherent. Magnification: X 190,000. FIG. 5. Infectious bronchitis virus after ether-Tween treat,ment for 12 hours. The membranes of many particles are intact and an indistinct internal component can be seen. Magnification: X 100,000. FIG. 6. A virus particle after treatment with 0.0170 sodium dodecyl sulphate. The surface projections are no longer present. Magnification: X 180,000.
sin-treated virus was examined therefore to determine whether any change in the surface could be seen. A O.l-ml volume of the virus concentrate
was incubated with 0.2 ml of freshly prepared 1%’ trypsin (Difco crystalline) for 35, 2, and 3 hours at 37°C. The morphology rernained normal, and spikes were still present.
STRUCTURE
OF INFECTIOUS
E$ect of Neuraminidase on, and Presence in, the Virus To test whether the presence of a mucoprotein inhibitor on the surface of the virus particle prevented haemagglutination under normal conditions, the virus was treated with neuraminidase and then examined for haemagglutinating activity. A l-ml volume of neuraminidase (Behringswerk) containing 200 units was incubated with 1 ml of the virus concentrate at 37°C. At 2 hours, 4 hours, and 16 hours, samples were taken and tested for haemagglutination at +4”C with 1% fowl red cells. No haemagglutination was observed. Subsequent electron microscopy on the samples showed no change in appearance from untreated controls. A sample of the virus concentrate was tested for the presence of neuraminidase by the method of Warren (1959). No neuraminidase was found in the sample. The estimations were kindly performed by Dr. J. Williams of the Protein Section, Department of Molecular Biology, Cambridge University. DISCUSSION
Our studies suggest that infectious bronchitis virus bears a superficial resemblance to the myxoviruses in that it is approximately the same size as influenza and carries projections on its surface. However, the proTABLE 1 -4 COMPARISON OF SOME PROPERTIES OF INFECTIOUS BRONCHITIS AND INFLUENZA Property Size Surface Internal component Ether
Haemagglutination NeuraminiNucleoprotein
Infectious
bronchitis
800-1200 A Pear shaped jections Not clarified
Influenza 800-1200 A Straight projectiona 90-100 A helix Complete disruption Rosettes and helix
pro-
Partial disruption of particles No rosettes or helix Not under normal conditions None found Ribonucleoprotein
-
Ribonucleoprotein
BRONCHITIS
VIRUS
jections are morphologically distinct from those of influenza and any other known myxovirus. Furthermore although both viruses are sensitive to ether, such treatment of infectious bronchitis does not result in the formation of rosettes from the surface projections. This is in contrast to findings with influenza virus of Hoyle et al. (1961). Table 1 summarises the main distinctions between infectious bronchitis and the myxoviruses. Infectious bronchitis thus shows certain unique features morphologically as well as biologically, and recognition of its true position in virus classification must wait until further information is forthcoming. REFERENCES ANDREWES, C. H., and HORSTMANN, D. M. (1949). The susceptibility of viruses to ethyl ether. J. Gen. Microbial. 3, 290-297. BEACH, J. R., and SCHALM, 0. W. (1939). A filterable virus, distinct from that of laryngotracheitis, the cause of a respiratory disease of chickens. Poultry Sci. 15, 199-215. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103-110. CORBO, L. J., and CUNNINGHAM, C. H. (1959). Haemagglutination by trypsin modified infectious bronchitis virus. Am. J. Vet. Res. 20, 876-883. DOMERMUTH, C. H., and EDWARDS, P. F. (1957). Electron microscope observations of thin sections of chorio-allantoic membranes infected with the virus of avian infectious bronchitis. J. Inject. Diseases 100, 74-81. HOYLE, L., HORXE, R. W., and WATERSON, A. P. (1961). The structure and composition of the myxoviruses. II. Components released from the influenza virus particle by ether. Virology 13, 448-459. LAVER, W. G. (1963). The structure of influenza virus. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology 20, 251-262. REAGAN, R. L., and BRUECKNER, A. L., (1952). Electron microscope studies of four strains of infectious bronchitis virus. Am. J. Vet. Res. 13, 417-418. REAGAN, R. L., HANSON, J. E., LILLIE, M. G., and CRAIGE, A. H., JR. (1948). Elert,ron micrograph of the virus of infectious bronchitis of chickens. Cornell Vet. 38, 190-191. WARREN, L. (1959). The thiobarbituric acid’ assay of sialic acids. J. Biol. Chem. 234, 1971-1975.
23, 403407
(1964)
The
Structure
of
Infectious
Bronchitis
Virus
D. M. BERRY Glaxo Research,
Greenford,
Middlesex,
England
J. G. CRUICKSHANK Department
of Pathology,
H. I’. CHU School of Veterinary
Cambridge AND
University,
England
R. J. H. WELLS
Medicine, Cambridge University, Accepted April 6, 1964
England
The structure of infectious bronchitis virus of chickens has been examined by the negative staining technique in the electron microscope. The particles are between 800 and 1200 A in diameter and bear projections on their surfaces. The effects of ether, sodium dodecyl sulphate, trypsin, and neuraminidase on the morphology and on certain biological properties of the virus were also examined. INTRODUCTION
Studies on the virus particle of infectious bronchitis of chickens have been few. The virus passes Berkefeld filters, grades V, N, and W (Beach and Schalm, 1939), but no accurate measurements of particle size have been made by filtration technique. Electron microscope studies on unstained and on shadowed preparations reveal spherical and “tadpole shaped” particles between 60 and 120 rnp in diameter (Reagan et al, 1948; Reagan and Brueckner, 1952). Chu (1961, unpublished work) demonstrated spherical particles in shadowed preparations apparently containing an outer capsule. In ultrathin sections of virus-infected chorioallantoic membranes, Domermuth and Edwards (1957) found numerous intracytoplasmic particles with dense outer portions and less dense centres giving a “doughnut” like appearance. Their particles were between 178 and 200 rnp in diameter. This paper reports high resolution electron microscope studies on the virus employing the negative staining technique of Brenner and Horne (1959). After the first figures of the virus had been obtained, attempts were made to split the particle to reveal
visible internal components. Various agents were used, and the particles were examined morphologically after each treatment. The structure of the treated particles was contrasted with the structure of a member of the influenza group of myxoviruses similarly treated. Further, as treatment of the particles with various agents may alter the surface properties of the virus, attempts were made to relate such alterations to changes in the morphology of the particles. For example, Corbo and Cunningham (1959) described haemagglutination of chicken erythrocytes by trypsin-treated virus, although under normal conditions infectious bronchitis does not haemagglutinate. MATERIALS
AND
METHODS
Four virus strains were examined. Two were strains isolated in England, Al63 by H. I’. Chu, and H17 by J. S. Garside. The other two strains employed in this study were the Connecticut strain and the Beaudette strain. All were cultivated in embryonated eggs by allantoic inoculation, harvests from which contained about lo6 EIDgO virus per milliliter. These virus harvests were con403
centrated by ultrac&rifugation for 1 hour at 54,000 9, aud the resulting pellets mere Wsuspended in 1 ‘/;i ammoniunl acetate. &asured portions were mixed with equal portions of 2 % potassium phosphotungstate (adjusted to pH 7.0 with 1% KOH) and t#he mixtures were either sprayed (with a Vaponephrin nebuliser) or dropped onto carboncoated grids. The grids were examined in the Siemens Elmiskop microscope at instrumental magnifications of 40,000 and 80,000 times with double condenser illumination. RESULTS
The virus was &bed for sensitilyity to c>t’her by the technique of Andrcwes and Horstrnann (19-19), and was found t,o be cthcr sensitive. For nlorphological studies a suspension of the egg-grown virus was shaken with an equal volume of ether overnight at room temperature. The ether was blown off with nitrogen, and the preparation was prepared as described above for microscopy. Particles were still recognisable and some retained some of their spikes, though most were partly disrupted. The damaged coats split open rather than disintegrated. The contents of some particles were released, but no morphological pattern could be made out (Fig. 5).
The particles from all four strains studied closely resembled each other. 11oderate numbers of particles were seen lying singly or in small clumps. The particles Effect of Xodium Dodecyl Sulphate showed considerable pleomorphism although Sodium dodecyl sulphate was used to try most of them were approximately circular in to split the virus, as Laver (1963) reported outline with diameters between 800 and 1200 the effectiveness of this agent in breaking up A (Fig. 1). Most but not all of the particles influenza particles. had projections from their surfaces. These A drop of virus concentrate was placed on “spikes” were often seen over part of the sur- a grid and fixed in formaldehyde vapour for face only and were less densely packed than 15 minutes. A drop of either 0.1% or 0.01% those seen in influenza viruses. They varied sodium dodecyl sulphate was placed on the considerably in shape. Commonly they ap- grid for 2 minutes and then blotted off. A peared to be attached to the virus by a very drop of 2% phosphotungstate was then put narrow neck and to thicken towards their on the grid for 2 minutes, and the excess distal ends, sometimes forming a bulbous fluid was drained off. mass 90-110 A in diameter (Figs. 2 and 3). Solutions containing 0.01% of the deterThese pear-shaped masses could sometimes gent removed all the spikes, but otherwise be seen lying on the surface of particles, ap- had no effect on the integrity of the parparently in clusters. In other particles the ticles (Fig. 6). spikes were more rod-shaped, but many were Solutions containing 0.1% of the deterbent in the middle. Spontaneous disruption gent produced partial or complete disruption was sometimes seen. That release of the in- of all virus particles. The coats appeared to ternal component can take place without break up into smallish pieces, but no rolling much damage to the outer coat is seen in up into rosettes was seen. Fig. 4, which shows an empty viral coat still In view of the removal of the spikes by lined with spikes. A proportion of the par- 0.1% sodium dodecyl sulphate, the haemagticles had invaginations in their centres. glutinating activity of the treated material Some of these were due to pooling of phos- was investigated using fowl erythrocytes. So photungstate on the loose outer coat, but in haemagglutinating activity could be demonothers the invaginations were well defined strated in sodium dodecyl sulphate-treated and were lined with a conspicuous mem- particles. brane. It was not clear whether the ringEflect of Trypsin shaped particles were truly of that shape or Haemagglutination of chicken erythrowhether the appearance was due to infolding cytes by trypsin-treated virus has been reof the membrane. ferred to above, and the morphology of tryp-
STRUCTURE
OF INFECTIOTJS
BRONCHITIS
VIRUS
405
FIG. 1. Infectious bronchitis virus showing the whole particles with the surface projections. Magnification: X 130,000. FIG. 2. A single infectious bronchitis virus particle showing the bulbous appearance of the surface projections. Magnification: X 200,000. FIG. 3. A particle of fowl plague virus (influenza A) showing the close-set cylindrical surface projections. Magnification: X 200,000.
FIG. 3. Two virus particles, one showing an intact membrane to which the projections remain adherent. Magnification: X 190,000. FIG. 5. Infectious bronchitis virus after ether-Tween treat,ment for 12 hours. The membranes of many particles are intact and an indistinct internal component can be seen. Magnification: X 100,000. FIG. 6. A virus particle after treatment with 0.0170 sodium dodecyl sulphate. The surface projections are no longer present. Magnification: X 180,000.
sin-treated virus was examined therefore to determine whether any change in the surface could be seen. A O.l-ml volume of the virus concentrate
was incubated with 0.2 ml of freshly prepared 1%’ trypsin (Difco crystalline) for 35, 2, and 3 hours at 37°C. The morphology rernained normal, and spikes were still present.
STRUCTURE
OF INFECTIOUS
E$ect of Neuraminidase on, and Presence in, the Virus To test whether the presence of a mucoprotein inhibitor on the surface of the virus particle prevented haemagglutination under normal conditions, the virus was treated with neuraminidase and then examined for haemagglutinating activity. A l-ml volume of neuraminidase (Behringswerk) containing 200 units was incubated with 1 ml of the virus concentrate at 37°C. At 2 hours, 4 hours, and 16 hours, samples were taken and tested for haemagglutination at +4”C with 1% fowl red cells. No haemagglutination was observed. Subsequent electron microscopy on the samples showed no change in appearance from untreated controls. A sample of the virus concentrate was tested for the presence of neuraminidase by the method of Warren (1959). No neuraminidase was found in the sample. The estimations were kindly performed by Dr. J. Williams of the Protein Section, Department of Molecular Biology, Cambridge University. DISCUSSION
Our studies suggest that infectious bronchitis virus bears a superficial resemblance to the myxoviruses in that it is approximately the same size as influenza and carries projections on its surface. However, the proTABLE 1 -4 COMPARISON OF SOME PROPERTIES OF INFECTIOUS BRONCHITIS AND INFLUENZA Property Size Surface Internal component Ether
Haemagglutination NeuraminiNucleoprotein
Infectious
bronchitis
800-1200 A Pear shaped jections Not clarified
Influenza 800-1200 A Straight projectiona 90-100 A helix Complete disruption Rosettes and helix
pro-
Partial disruption of particles No rosettes or helix Not under normal conditions None found Ribonucleoprotein
-
Ribonucleoprotein
BRONCHITIS
VIRUS
jections are morphologically distinct from those of influenza and any other known myxovirus. Furthermore although both viruses are sensitive to ether, such treatment of infectious bronchitis does not result in the formation of rosettes from the surface projections. This is in contrast to findings with influenza virus of Hoyle et al. (1961). Table 1 summarises the main distinctions between infectious bronchitis and the myxoviruses. Infectious bronchitis thus shows certain unique features morphologically as well as biologically, and recognition of its true position in virus classification must wait until further information is forthcoming. REFERENCES ANDREWES, C. H., and HORSTMANN, D. M. (1949). The susceptibility of viruses to ethyl ether. J. Gen. Microbial. 3, 290-297. BEACH, J. R., and SCHALM, 0. W. (1939). A filterable virus, distinct from that of laryngotracheitis, the cause of a respiratory disease of chickens. Poultry Sci. 15, 199-215. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103-110. CORBO, L. J., and CUNNINGHAM, C. H. (1959). Haemagglutination by trypsin modified infectious bronchitis virus. Am. J. Vet. Res. 20, 876-883. DOMERMUTH, C. H., and EDWARDS, P. F. (1957). Electron microscope observations of thin sections of chorio-allantoic membranes infected with the virus of avian infectious bronchitis. J. Inject. Diseases 100, 74-81. HOYLE, L., HORXE, R. W., and WATERSON, A. P. (1961). The structure and composition of the myxoviruses. II. Components released from the influenza virus particle by ether. Virology 13, 448-459. LAVER, W. G. (1963). The structure of influenza virus. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology 20, 251-262. REAGAN, R. L., and BRUECKNER, A. L., (1952). Electron microscope studies of four strains of infectious bronchitis virus. Am. J. Vet. Res. 13, 417-418. REAGAN, R. L., HANSON, J. E., LILLIE, M. G., and CRAIGE, A. H., JR. (1948). Elert,ron micrograph of the virus of infectious bronchitis of chickens. Cornell Vet. 38, 190-191. WARREN, L. (1959). The thiobarbituric acid’ assay of sialic acids. J. Biol. Chem. 234, 1971-1975.