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CENTRIFUGATION STUDIES
OF INFECTEOUS BRONCHITIS VIRUS

 

Thesis gov Hm Degree o§ p5. D.
MICHIGAN STATE UNIVERSITY

Lee Floyd Eliis
1965

 

THflfls

LIBRARY

_ Michigan Statue
University

 

This is to certify that the

thesis entitled

"Centrifugation Studies of Infectious
Bronchitis Virus"

presented by

Lee Floyd Ellis

has been accepted towards fulfillment
of the requirements for

ELL—degree inflQQLLOJ-Ogy 5‘
Public Health

MimZ/dmwk

Major professor

 

0-169

ABSTRACT
CENTRIFUGATION STUDIES OF INFECTIOUS BRONCHITIS VIRUS
by Lee Floyd Ellis

The plaques produced by infectious bronchitis virus
(IBV), Beaudette strain", in chicken embryo kidney cell (CEKC)
cultures vary from 1 to 7 mm in diameter. Medium plaques, 3
to 5 mm, are produced most frequently, but smaller plaques
are present at limiting dilutions of viral infectivity and in
the presence of small amounts of antiserum. Two pOpulatiOns
in this strain are suggested by differential thermostability
of virus cultivated in embryonating chicken eggs and by DEAE
separation of the mixture into small and large plaques on
CEKC cultures. Therefore, attempts were made to ascertain
.if separable pOpulations occurred in CEKC cultured virus
re3ponsible for the different sized plaques and to determine
some of their physical prOperties.

Isopycnic density gradient centrifugation with cesium
chloride was employed in an attempt to separate the virus
pOpulation. Cesium chloride accelerated the inactivation of
virus in solution and was toxic to the cell cultures at low
dilutions. Maximum virus infectivity was at 1.24 Specific
gravity, but ultraviolet absorbance was not associated with
this band. The virus suSpension could be clarified by passage
through a Sephadex G-ZOO column followed by differential

centrifugation to remove the ultraviolet absorbing components

of the medium. Only small amounts of infective virus were
recovered.

Linear sucrose gradients employed for both zonal and
isopycnic density gradient centrifugation markedly improved
the recovery of infective virus as compared to cesium chloride
gradients. The virus was homogeneous according to these
procedures, but the Specific gravity of the individual infec-
tive viral units was diverse. Only small differences between
individual plaque isolates were detected. Maximum infectivity
was associated with 1.19 specific gravity solution. Differen-
tial centrifugation clarified the infective virus suSpenSionS
of ultraviolet absorbing components due to the culture medium
and no ultraviolet absorbance was associated with the infective
virus band.

The sedimentation coefficient of virus purified by a
sucrose gradient was 334 S as determined by a partition cell
method of centrifugation. From the S value, the Sphere is
calculated to be approximately 80 mu diameter as compared to
100 mu by electron microscOpy.

From the results obtained, the Beaudette strain of IBV
is considered to be a homogeneous p0pulation with reSpect to
the physical characteristics reported. The Specific gravity
of individual infective viral units is variable and is con-
sidered one characteristic of IBV. The differences in the
Size of the plaques produced were not attributable to dif-

ferent pOpulations.

CENTRIFUGATION STUDIES
OF INFECTIOUS BBONCHITIS VIRUS

By

Lee Floyd Ellis

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

oocroa or PHILOSOPHY

Department of Microbiology and Public Health
1965

ACKNOWLEDGEMENTS

The author wishes to express his appreciation to Dr.
Charles H. Cunningham for his guidance and inSpiration and
to Dr. Jack J. Stockton and Dr. Harold L. Sadoff for their
guidance and encouragement throughout this graduate program.
Appreciation is also eXpressed to my wife and family for
their forebearance of a part time husband and father during
this time.

Acknowledgement is also made to Mrs. Martha P. Spring
and my fellow graduate students for their interest and help.
I wish to thank Mr. Gordon Spink for his help with the elec-
tron microscOpy.

This study was supported in part by the Michigan Agri-

cultural Experiment Station.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . .

Infectious Bronchitis Virus . . .

Density Gradient Centrifugation .

Sedimentation Velocity Centrifugation

MATERIALS AND METHODS . . . . . . . . .
Cell Culture . . . . . . . . . . .
Stock Virus . . . . . . . . . . .
Plaque Isolates . . . . . . . . .
Plaque Assay . . . . . . . . . . .
Cesium Chloride Density Gradients
Sucrose Density Gradients o . . .
Sedimentation Velocity . . . . . .
Electron MicroscOpy . . . . . . .

RESUDTS . . . . . . . . . . . . . . . .
Virus Plaques . . . . . . . . . .
Differential Centrifugation . . .
Cesium Chloride Density Gradients
Sephadex Chromatography . . . . .
Zonal Centrifugation . . . . . . .
Sucrose Density Gradients . . . .

111

Page
ii

vii

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k‘ r4 F‘ t4 F4 l4 r4 l4 P“ i4
c- u: \n n) to a) to F‘ r4 <3

Electron MicrOSCOpy . . .

Sedimentation

Tables . .
Figures . .
DISCUSSION . . .
SUMMARY . . . .
LITERATURE CITED

Coefficients

iv

Page
14
15
18
20
31
37
38

Figure

1.

LIST OF FIGURES

Isopycnic density gradient centrifugation of
extracellular IBV in cell culture medium.

Virus not clarified of concentrated. 2.5 M
0301, 117,500 X g, 0 C, 36 hr. . . . . . . . .

ISOpycnic density gradient centrifugation of
cell culture medium. 2.5 M CsCl, 117,500 X g,
0 C, 36 hr. 0 O 0 O O O O O O O O O O O O O O

Isopycnic density gradient centrifugation of
IBV. Extracellular virus in cell culture
medium clarified by Sephadex G-200 and centri-
fugally concentrated. 2.5 M CsCl, 117,500 X g,
0 C, 36 hr. 0 O O O O O O O O O O O O O O O C

Isopycnic density gradient centrifugation of
extracellular virus in cell culture medium.
Virus not clarified or concentrated. 2.0 M
CsCl, 117,500 X g, 0 C, 24 hr. . . . . . . . .

Column chromatography with Sephadex G-200 of
extracellular IBV in cell culture medium. . .

Zonal centrifugation of extracellular IBV in
cell culture medium, not clarified or concen-
trated. Linear sucrose gradient, 100,000 X g,
0 C, 3 hr. 0 O O O O O 0 O O O O O 0 O O O O 0

ISOpycnic density gradient centrifugation of
IBV. Extracellular virus in cell culture
medium clarified and concentrated by differ-
ential centrifugation. Linear sucrose A
gradient, 124,000 X g, 1 C, 12 hr. . . . . . .

Isopycnic density gradient centrifugation of
IBV. Maximal infectivity fractions from
previous density gradient (Figure 7) combined
and then concentrated by centrifugation.
Linear sucrose gradient, 124,000 X g, l C,

12 hr. 0 O O O O O O I O O O O O O O O O O O O

Page

20

21

22

23

24

25

26

27

Figure
9.

10.

11.

Page

ISOpycnic density gradient centrifugation of
extracellular IBV in cell culture medium. Iso-

lates from fraction 5, 9, and 13 on zonal
centrifugation tube (Figure 6). Comparison

of virus on linear sucrose gradient, 124,000 X g,

l C, 12 hr. 0 O O O O O O O O O O O O O O O I O O 28

Isopycnic density gradient centrifugation of
intracellular IBV in cell culture medium,

not clarified of concentrated. Linear

sucrose gradient, 124,000 X g, l C, 12 hr. . . . . 29

Electron micrograph of infectious bronchitis

virus from CEKC purified by iSOpycnic density
gradient centrifugation. Virus dissolved in

1 per cent ammonium acetate and stained with
phOSphotungstate. Magnification X 120,000. . . . 30

vi

LIST OF TABLES
Table

1. Differential centrifugation of extracellular
IBV in cell culture medium. Centrifuged for
1 hour in angle head rotor, 5 C. . . . . .

2. Sedimentation coefficients of infectious
bronchitis virus, Beaudette Strain.

vii

Page
0 O 18
. . 19

INTRODUCTION
The Beaudette strain of infectious bronchitis virus
(IBV) causes plaques in chicken embryo kidney cell cultures
(CEKC) providing a biological assay method for this virus.
The objective of this study was to determine physical character-

istics of the virus by centrifugation.

LITERATURE REVIEW
Infectiousggronchitis Virus

Infectious bronchitis virus is the etiological agent of
a world wide, Specific, highly contagious respiratory disease
limited to chickens. The Beaudette embryo-adapted strain
readily kills 10-day-old chicken embryos (Beaudette and
Hudson, 1937; Delaplane and Stuart, 1941). It can also be
cultivated in the isolated chorioallantoic membrane (Cunningham,
1960), and in chicken embryo liver cell culture (Fahey and
Crawley, 1956). When adapted to chicken kidney cell cultures
(CEKC), microscopic cytopathic effects and macroscopic plaques
are produced (Spring, 1960).

The usual viral replication cycle occurs with IBV. After
inoculation, a 4 hour eclipse phase was followed by a rapid
increase of cell associated and released virus. Released virus
continued to increase exponentially during the next 30 hours.
Acridine orange staining of the infected cell showed accumula-
tion of RNA in the cytoplasm. Syncytia were formed during the
first 24 hour post inoculation, but were absent thereafter
(Akers, 1963).

Infectious bronchitis virus exists in two phases: the
thermostable original (0) phase as originally isolated from
infected chickens is antigenic and pathogenic to chickens; the
thermosensitive derivative (D) phase develops as the result of
passage in chicken embryos. The 0 phase can be selected from
an 0-D phase mixture by differential heat inactivation of the

-2-

 

It?! 1?:

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ta

de

the

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-3-
D phase at 56 C (Singh, 1960).

The virus is a Sphere having a diameter of 60 to 80 mu
(Nazerian, 1960), 120 mu (Buthala, 1956) or 80 to 120 mu
(Berry et a1., 1964) according to electron microscoPy; it has
a density of 1.15 in sucrose (Buthala, 1956) and 1.23 in
cesium chloride (Tevethia, 1964); it elutes from diethylamino-
ethyl cellulose (DEAE) at 0.45 and 0.90 M NaCl, pH 7.2.

Direct agglutination of chicken erythrocytes occurs only after
treatment of the virus with trypsin (Corbo and Cunningham,
1959). Horse erythrocytes modified with tannic acid can be
'used for an indirect hemagglutination test for IBV (Brown

et a1., 1962). The virus is ether sensitive (Akers, 1963;
.Mohanty and Chang, 1963; Berry et a1., 1964).

Density Gradient Centrifugation

Centrifugal force is used to determine the hydrated size,
buoyant density, and molecular weight of macromolecules. The
rate of sedimentation of a solute can be determined with either
an analytical ultracentrifuge using an optical system or a
preparative centrifuge using density gradients and subsequent
analysis of the fractions to determine the position of the
solute. Solutions of sucrose, CSCl, RbCl, glycerol, potassium
tartrate, albumin, Cssou or LiCl are commonly used to form the
density gradients.

Density gradient centrifugation is classified into three
types. First, for stabilized moving boundary centrifugation,
the solute is placed on a preformed gradient. After centrifu-

gation the fractions are collected and the positions of the

-4-
boundaries are determined from which sedimentation coefficient
of each boundary is calculated.

Second, the zonal centrifugation is used to purify
materials. For this procedure the solute is layered on a pre-
formed gradient and after a short period of centrifugation, the
fractions are collected and the positions of the boundaries
are determined. Macromolecules of equivalent sedimentation
coefficients form boundaries within the gradient.

The third type, iSOpycnic density gradient centrifuga-
tion, separates particles according to their buoyant density.
The solute can either be layered on or mixed into the gradient
material and then centrifuged until the solute attains equili-
brium. The contents are then fractionated and the position
of the bands determined.

The specific gravity of the agents for the following
viral diseases has been determined: measles, 1.25, (Norrby,
1964); Newcastle disease (NDV) from avian cells, 1.219 to
1.221, and from mammalian cells, 1.236 to 1.242, (Stenback
and Durand, 1963); Rous sarcoma, 1.16 to 1.19, (Crawford, 1960);
polyoma "full" particles, 1.339, "empty" particles, 1.297, (Abel
and Crawford, 1964); simian virus 40 (SV 40), ”full” particles,
1.32, “empty“ particles, 1.29, (Black et a1., 1964); Sindbis,
1.21, (Pfefferkorn and Hunter, 1963); infectious vesicular
stomatitis, 1.20, (Prevec and Whitmore, 1964); adenovirus type
2, 1.34 (Green and Pina, 1963); influenza PR 8, 1.18, (Lauffer
and Stanley, 1944); and herpes simplex, 1.26 to 1.275, (Roizman
and Roane, 1961).

-5-

Cellular material may be incorporated into some viral
particles (Franklin, 1962). The findings of radioactive
labeled phospholipid of cellular origin in Sindbis virus
(Pfefferkorn and Clifford, 1964), density variation of NDV
in avian and mammalian cells (Stenback and Durand, 1963), and
the density range of Rous sarcoma virus (Crawford, 1960) all
suggest incorporation of cellular material in the virus.

Other virus fractions separable by density gradient cen-
trifugation are hemagglutination activity from measles virus
(Norrby, 1964); non-infectious Sindbis virus (Prevec and
Whitmore, 1963); and precipitating antigens of IBV (Tevethia,
1964).

Sedimentation Velocity Centrifugatign

The sedimentation coefficient (S) of many proteins and
viruses have been determined. The light adsorption and refrac-
tive methods of analysis are often not applicable in biology
because concentration of the solute is too low. But with
known materials having 4-2000 S values, comparable results were
obtained by preparative ultracentrifugation and other physical
methods (Hogeboom and Kuff, 1954; Martin and Ames, 1961; Polson
and van Regenmortel, 1961).

The S value of the agents for the following viral diseases
has been determined: polyoma ”full” particles, 238 S, and
"empty” particles, 140 S (Crawford and Crawford, 1963); SW 40,
242 S (Winocour, 1963); papilloma ”full” particles, 298 S and
"empty" particles, 172 S (Crawford and Crawford, 1963); influenza
A 700 S (Friedewald and Pickels, 1944); influenza PR 8, 722 S

-5-
(Lauffer and Stanley, 1944); poliovirus type 2, 165 S (Polson
and van Regenmortel, 1961); turnip yellow mosaic, 105 S (Polson
and van Regenmortel, 1961); barley stripe mosaic, 182-206 S
(Brakke, 1958); wheat streak mosaic, 159-173 S (Brakke, 1958);
brome mosaic, 75-83 S (Brakke, 1958); potato yellow-dwarf,

810-950 S (Brakke, 1958).

MATERIALS AND METHODS

Cell Culture

Chicken embryo kidney cell cultures (CEKC) were prepared
as described by Cunningham (1963) and Cunningham and Spring
(1965). The kidneys from 16- to l7-day-old chicken embryos
were removed aseptically, washed with Hanks' balanced salt
solution (B33) and cut into 1 to 2 mm pieces. Blood clots and
debris were removed and the fragments of kidneys were then
transferred by a pipette to a 500 m1 fluted flask containing a
Teflon-covered magnetic bar. Ten m1 of 0.25 per cent trypsin
(1:250)* in BSS, pH approximately 8.0, was added per pair of
kidneys. The contents were stirred with a magnetic stirrer for
1 hour at room temperature or for 30 minutes at 37 C. The
cell suSpenSion was filtered through 2 layers of cheese cloth
and then centrifuged at 400 X g for 5 minutes at 4 C. The
supernatant fluid was decanted and the packed cells were resus»
pended in BSS. Washing was repeated twice.
7

Packed cells were resuSpended to 10 cells per ml in the
cultural medium consisting of medium 199 (Morgan et a1., 1950)
supplemented with vitamins, amino acids and L-glutamine in the
concentration in Eagle's basal medium (Eagle, 1959), 5 per cent
newborn calf serum, 100 units penicillin, 0.1 mg dihydrostrepa
tomycin and 100 units Mycostatin per m1, reSpectively, The

suspension was filtered through two layers of gauze and 4 ml

was diSpensed per 15 x 60 mm plastic tissue culture Petri

 

*
Difco Laboratories, Detroit, Michigan
-7-'

-8-

plate. Incubation was at 37 C, in 8 per cent C02 and 80-85
per cent relative humidity with 2 atmOSpheric changes per hour.
A satisfactory monolayer of cells was formed after 44 to 60
hr.
Stock Virus

The 30th and 100th passages of the Beaudette culture of
IBV (North Central Repository Code Number IBV 42) adapted to
CEKC were used (Cunningham and Spring, 1965). The inoculum
per cell culture was 0.1 ml of undiluted extracellular fluid
containing the virus. Forty-eight hours after inoculation,
the cultural medium containing extracellular virus was sub-
jected to low Speed centrifugation to remove any cells. Five
m1 portions of the supernatant fluid were placed in vials for
storage at -62 C. The titer of the 3lst passage of virus was

2 X 106 PFU per m1 and the titer of the lOlst passage of virus

was 6 X 10“

PFU per m1.

To obtain the cell associated virus, the monolayers were
washed 4 times with B83 20 hr following inoculation and then
suspended in cultural medium. The cells were ruptured by sonic
vibration at 20 kc for 2 minutes. After low Speed centrifuga-
tion to remove cells and debris, 5 m1 portions of the superna-
tant fluid were placed in vials for storage at -62 C.

Plague Isolates

Two to three days after inoculation unstained plaques were
visible by indirect light. Two mm diameter glass tubing in
three inch lengths was thrust through the center of a plaque

to the plate. The small core was removed and transferred into

-9-
2 m1 of cultural medium which was stored at -62 C.
Plaque Assay

Cell cultures were washed twice with 2 m1 of B88 and then
inoculated with 1.0 m1 of an apprOpriate dilution of virus in
BSS. With the density gradient studies, only one culture was
used for each of the serial ten-fold dilutions. Since 20 or
more fractions were assayed, it was not feasible to prepare
and use more cultures per dilution. For the sedimentation
velocity studies, 4 cultures were used for each of the serial
ten-fold dilutions. After an adsorption period of 60 to 120
minutes at 37 C, the inoculum was poured off and the cells were
overlaid with 4 m1 of 0.9 per cent Difco Noble agar in cultural
medium. On the 3rd or 4th day the cells were stained with 0.1
per cent neutral red in B88 and the plaques were counted. One
plaque forming unit (pfu) is that amount of virus causing the
formation of a single plaque.
Cesium ChlorideAQensity Gradients

For iSOpycnic (equilibrium) density gradient centrifugam
tion (Meselson, et a1., 1957) the SW 39 swinging bucket rotor
in a Model L preparative ultracentrifuge (Beckman Instruments
Inc., Spinco Division) was used. One ml of virus suspension
was mixed with 4.0 m1 of 2.0 or 2.5 M 0801 in 0.05 M Tris bufm
fer*, pH 7.4, in lusteroid tubes. A control tube was prepared
in the same manner using culture medium. The mixtures were
centrifuged at 100,000 to 125,000 X g for 20 to 36 hours and
allowed to decelerate without braking. The bottom of the tube

was pierced with a double-beveled 26 gauge needle. Four drop

-10-
fractions were collected and the Specific gravity was deter-
mined with a tared 25 uliter micropipette.

The volume of each fraction was adjusted to 2.0 ml with
BSS containing 0.01 per cent casein hydrolysate*. The absor-
bance at 260 mu and 280 mu was determined with a Beckman DB
Spectrophotometer. Viral infectivity was assayed, according
to the method previously described, immediately after centri-
fugation because the virus was relatively unstable. Dilutions
of 1/200 or greater were used because CSCl was toxic to the
cells.
Sucrose_QenSityfGradients

Linear gradients were prepared using a Lucite block with
two cylindrical chambers interconnected by a small channel
and a drain tube in the bottom of one chamber (Hogeboom and
Kuff, 1954). For isopycnic density gradient centrifugation,
thirty per cent sucrose in Tris buffer, pH 7.4, was placed
in the chamber without the drain tube. Sixty per cent sucrose
in Tris buffer, pH 7.4, was placed in the other chamber. Equal
quantities of resuSpended virus were added to each chamber.
The contents of the latter chamber were stirred with a small
paddle. As the sucrose solutions drained into a lusteroid
centrifuge tube, a linear gradient from 1.13 to 1.25 specific
gravity was formed. The centrifuge was Operated at 125,000 X g.

For zonal centrifugation, the procedure was the same except the

 

*Sigma 121, tris (hydroxymethyl) aminomethane, Sigma Chemical
Co., St. Louis, Missouri.

*Nz Amine Type E, Sheffield Chemical Co. Inc., Norwich, N.J.

-11-
sucrose concentrations were 20 and 40 per cent, resuSpended
virus was layered onto the preformed gradient and centrifuged
at 73,000 X g. The rotor was stepped without using the brake
and the fractions were collected as with the cesium chloride
gradients.

Sedimentation Velocity

Three ml of 30% sucrose was placed in duplicate centrifuge
tubes and was then overlaid with 2.0 m1 of virus suSpenSion
(approximately 10,000 pfu/ml) in 5 per cent sucrose. The
tubes were rotated gently to form a diffuse interface.- The
control tube for temperature determination consisted of the
sucrose solutions only. A cooled SW 39 rotor was used. After
centrifugation at 73,000 X g for varying periods, the uppermost
1.5 ml was carefully removed from each tube for assay of virus
as described previously. The temperature of the solution in
the control tube was determined immediately after centrifuga-
tion.
Electron Microscopy

Virus suspensions were subjected to 90,000 X g centrifugal
force and the pellets were resuSpended in 1 per cent ammonium
acetate, pH 7.0. Equal volumes of the virus and 1 per cent
phosphotungstic acid were mixed together and small amounts were
placed on Carbon coated grids. When dried sufficiently the

virus was examined in a Phillips Model 100 electron microscOpe.

RESUDTS

Virus Plaques

The majority of the discrete plaques were 3~5 mm in dia-
meter, but a few were 1-2 mm. The size of the plaques was not
significantly altered by: heating the virus suSpenSion at
50 C for 10 minutes; sonification at 20 kc for 2 minutes; vary»
ing the concentration of sodium bicarbonate from 0.05 to 0.20
per cent; or the use of cells from 40 to 68 hr old. Low con»
centrations of Specific antiserum in the agar overlay medium
decreased the size of the plaques formed and higher concentra-
tions prevented plaque formation.
Differential Centrifugation

The minimal centrifugal force to effectively sediment the
virus was 70,000 X g, based on the per cent recovery of infec-
tive virus and light absorbance at 260 mu and 280 mu (Table 1).
The high absorbance by the supernatant fluid was due to the
serum protein and phenol red in the cultural medium, whereas
the sediment was resuSpended in BSS without phenol red. No
explanation of the absorbance changes of the supernatant fluid
is offered.
Cesium ChlorideQensityGradients

The number of fractions (four drops) collected from the
gradient tubes varied from 17 to 23 fractions. The total volume
was always 5.0 m1. Therefore, for uniformity of the results,
the fractions were plotted according to their volume.

Using 2.5 M cesium chloride gradients with extracellular

112_

-13-
virus cultures, absorbance bands (concentrated zone of material)
were at 1.32 and 1.26 specific gravity (Figure 1). Similar

bands were present in gradients of control culture medium

(Figure 2). After passage of extracellular virus culture through
Sephadex G~200 and concentrated by centrifugation, the 1.32
specific gravity band material was not present (Figure 3).

When a 2.0 M cesium chloride gradient was used, only an absora
bance band at 1.22 specific gravity was present from the extra»
cellular virus culture (Figure 4).

With either 2.0 or 2.5 M cesium chloride gradients, the
infective virus from the unclarified culture was in a broad
band at 1.24 Specific gravity (Figure 1 and 4). From the
clarified virus culture, the infective virus was in a broad
band at 1.20 specific gravity (Figure 3). Most fractions pro-
duced large and small plaques regardless of the Specific
gravity.

Sephadex Chromatography

Infective virus was at the leading edge of the excluded
material eluting from Sephadex G-200 (Figure 5). Other com»
ponents from the cell culture medium were also in these frac-
tions. Phenol red from the culture medium was eluting in
fraction 15 through 21. Only 2.2 per cent of the infective
virus was recovered.

Zonal Centrifugatign

One band of infective virus migrated into the sucrose

gradient and ultraviolet absorbance was not associated with

this band (Figure 6). Most of the ultraviolet absorbing comm

-14-
ponents did not migrate in the centrifugal field.
Sucrose Density Gradients

Virus cultures clarified by differential centrifugation
had one broad band at 1.19 specific gravity with some virus
distributed throughout the gradient (Figure 7). When the
four fractions containing most of the virus from the first
sucrose gradient were combined and used in a second sucrose
gradient, only one broad band around 1.19 specific gravity
remained (Figure 8).

Plaque isolates from fraction 5, 9 and 13 of the zonal
centrifugation gradient were prepagated in CEKC and the culture
medium harvested at 48 hr. Each isolate was then centrifuged
in a sucrose gradient (Figure 9). Isolates from fraction 5
and 9 were similar with reference to the broad diSpersion of
the infective virus and the maximum infectivity at 1.19 specim
fic gravity. The isolate from fraction 13 was more homogeneous
and the maximum infectivity was at 1.20 specific gravity.

With the cell associated virus, a broad area from 1.13
to 1.22 Specific gravity contained infective virus. This
indicates an inhomogeneous material. By indirect lighting a
diffuse band was observed in the upper part of the contents
of the tube.

Electron Microsc0py

Electron micrographs of the various preparations confirmed
the presence of virus in the infective fractions (Figure 11).
The virus particles were 100 to 120 mu in diameter and typically

possessed dark centers and a light exterior shell. Cell asso-

-15-
ciated virus preparations contained irregular shapes and forms
suspected of being viral particles.

Sedimentation Coefficient
The sedimentation coefficient (S) is the velocity of a
sedimenting molecule in a unit field and is defined by:
dx

 

w2 x dt
w angular velocity in radians per second

x distance in cm of the protein boundary from the
center of the axis of rotation

t time in seconds
The unit of sedimentation commonly employed is the Svedberg
(S) which is equivalent to 1 X 10"13 seconds. The S values
were calculated according to Polson and van Regenmortel, (1961).

x % a

 

3.5 n log
t x + a Vt

vo

 

nt viscosity of solvent at temperature of centrifugation
n20 viscosity of solvent at 20 C

x distance in cm from upper meniscus to center of
axis of rotation

a effective column length
vt virus titer after centrifugation
N revolutions per minute

T time of centrifugation of minutes

 

N16..

The following formula was used to convert the observed S

value to 820 w the equivalent sedimentation rate in water
9

at 20 C (Tanford, 1961).

 

S. . =

viscosity of medium at 20 C

viscosity of water at 20 C

partial Specific volume of the solute
specific gravity of medium at 20 C

specific gravity of water at 20 C

The specific gravity was determined with a ten m1

pycnometer and double distilled water was the standard

(Daniels et a1., 1956).

SEQ-'0

 

specific gravity of unknown solution
Specific gravity of water
weight of medium in pycnometer

weight of water in pycnometer

-17-

Viscosity of the solvent with double distilled water as

the standard was determined with a Ostwald viscometerc The

equation for calculation was (Glasstone and Lewis, 1960):

d
w

t d nW
tw dw

 

viscosity of solvent
viscosity of water

time observed for solvent to fall from one point to
another in the instrument

time observed for water to fall from one point to
another in the instrument

specific gravity of solvent

Specific gravity of water

The sedimentation coefficient of IBV, Beaudette strain

from CEKC, was 334 i #7 S as determined by this method (Table

12). The S values decreased with increasing time of centri-

fugation, but duplicate tube results complemented each othero

u

The initial virus concentration was approximately 1 X 10 pfu

per ml to reduce the possibility of clumpingo

Table lo Differential centrifugation of extracellular IBV
in cell culture mediumo Centrifuged for 1 hour in angle

head rotor, 5 Co

 

 

Centrifugal Optical Density Per Cent
Force Supernate Sediment Activity
x g 260mu 280mu 260mu 280mu Recovered
10,000 00340 00490 00030 00030 1.2
30,000 00350 09515 00045 00045 401
50,000 0°3u5 00520 09045 00045 #04
70,000 00355 Oofho 00085 00085 900
90,000 00355 00550 00080 00080 8.5

 

M18-

Table 20 Sedimentation coefficients of infectious bronchitis

virus, Beaudette straino

 

 

 

Time of Viscosity Virus Titer Vt S x 10*
Centrifu- Before After - ZO’W
gation V
o
6019 000109 18,200 6650 00365 382
8069 090109 18,200 2550 00140 382
13069 000109 18,200 620 00034 277
12019 000115 3,900 90 00023 324
12019 000115 3,900 340 00088 30“
Average 33“

soda #7

The effective column length was l018 cm, N was 30,000 rpm,
and x was 5066 cm0

M19“

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Figure 110 Electron micrograph of infectious bronchitis
virus from CEKC purified by isopycnic density gradient
centrifugationa Virus dissolved in l per cent ammonium acetate

and stained with phosphotungstate° Magnification X 120,000,

~30-

DISCUSSION

The Specific gravity of infectious bronchitis virus was
1022 to 1027 in CsCl and 1018 to 1020 in sucroseo Further
centrifugation did not decrease the band Widtho This band
width was not considered due to Brownian movement, since with
homogeneous materials the band width varies inversely to molew
cular weight (Meselson et alg, 1957)o The molecular weight
of IBV estimated from the S value, is 75,000,000, therefore,
the band width should have been very narrowo Heterogeneity
of the density of the hydrated particles is one possible
explanation of the broad bando Influenza virus PR 8 is homo»
geneous, but other influenza strains are heterogeneous regard~
ing density (Lauffer and Stanley, l9fl4)o The density of Rous
sarcoma virus ranged from 1016 to 1019 in rubidium chloride
(Crawford, 1.960)o Therefore, heterogeneous density may be
a characteristic of some viruses, eSpecialiy those maturing
at the cell surface such as IBVo

The heterogeneity of the density could be a result of
compositional differences among the particleso According to
Franklin (1962) the ribonucleic acid (RNA) content per infecw
tious unit of a myxovirus is variable and since the density
of RNA is approximately 109, a small compositional change
would markedly change the particle densityo Incorporation
of cellular phospholipid into Sindbis virus maturing at the
cell surface has been demonstrated by radioactive labeling
(Pfefferkorn and Clifford, 1961+)o Since IBV is inactivated

l3ll

-32-
by ether and sodium deoxycholate, it is assumed the outer
surface contains lipid (Akers, 1963; Mohanty and Chang, 1963;
Berry et a1., 1961+)o If varying amounts of this cellular
material were added to the virus particle, then its density
would be heterogeneouso The infective unit may be numerous
particles in a clump enclosed by a membranous material, and
very large clumps would have a higher density than smaller
clumpso These composition factors may account for Newcastle
disease virus (NDV) obtained from avian cells being less dense
than NDV obtained from mammalian cells (Stenback and Durand,
1963)o

Dehydration of IBV by the gradient solution would probably
effect the density and heterogeneity of the viral particle0
Maximum infectivity of IBV was at 1024 specific gravity in
CsCl and 1°19 specific gravity in sucrose; the molarity at
these points was 200 and 1025, respectively. The particles
would be more anhydrous in CsCl than in sucrose because of the
osmotic effecto With influenza virus PR 8, the density was
1010 in water, 1018 in sucrose and 1027 in the anhydrous state
(Lauffer and Stanley l9h4)o Regarding heterogeneity, the
density range of IBV was greater in CsCl than in sucrose, but
both solutions would be exerting osmotic influenceso The
molarity and size of the molecules of the solvents are different
and may affect the Speed of hydrationo If varying amounts of
extraneous protein material were enclosing the particles, the
amount of water contained by this protein could be changed

easily by the gradient solutionso

-33-

There was a low recovery of the infective virus by centria
fugationo These results may have been due to l) clumping of
the virus particles, 2) incomplete sedimentation, or 3) physi~
cal or chemical inactivation of the viruso The pellets from
a one hour cycle of differential centrifugation contained less
than 10 per cent of the infectivity of the original sampleo
The virus is stable for one hour at 5 C (Cunningham, 1960) so
the inactivation that occurred was probably not caused by
thermal influenceso Using IBV from allantcic fluid, Buthala
(1956) found only 10 per cent of the infective virus in pellets
after centrifugation for 3 hours at b0,000 rpm, but infective
virus was still present in the supernatant fluid. In the
present work with IBV from CEKC, a cushion of high density
sucrose increased the yields in the sedimented layer two-foldo
The decrease in infectivity was accelerated by 0801, whereas
sucrose protected the viruso These differences are reflected
by the recovery rates from the gradients: 802, 19.3 and 204
per cent for sucrose, and 107, #00 and 1500 per cent for CsClo
Disaggregation of virus clumps could account for the 204 per
cent recovery in one case, since only one plaque deve10ps
regardless of the number of particles initially infecting a
small areao The above evidence supports either the clumping
or inactivation explanations for the low recoveryo

There is a high degree of precision and reproducibility
for assay of the virus by the enumerative dose reSponse under
carefully controlled conditions (Cunningham and Spring, 1965)o

Primary cell cultures, because of their lack of synchronous

-34-

growth, introduce a variable into the bioassay. Some of the
variance in this study was from using single plates in the
bioassay, but the large number of fractions prohibited dupli—
cation. Recently Homma and Graham (1965) reported that intact
mengo virus penetrated the cell membrane, but the protein coat
of the virus was not subsequently removed to release the infecu
tive RNA. Since the ratio of particles per infective unit is
usually 100 or greater, small changes affecting this ratio
would markedly influence the number of infective units.

The diameter of IBV depends on the method of measurement,
the source of virus, and procedures for preparation. By cal-
culation from sedimentation velocity values, the diameter of
the hydrated particle is 56 mu if the density is 1.19 and
80 mu if the density is 1.10. Assuming the density of IBV in
water to be 1.10, the same as influenza virus, then 80 mu is
a reasonable estimate and agrees with electron micrographs of
IBV that show 100 mu particles. Since some virus was sedi-
mented at 10,000 X g part of the infectious material must have
been in large clumpso This was confirmed by electron micro«
scopy° Similarly, clumps of SV #0 enclosed in a membrane are
sedimentable at 8,000 rpm (Black et a1., 196h).

Infectious virus did not account for the visible bands,
but these bands were partially identified. The 1.32 Specific
gravity band in CsCl gradients may be caused by the protein
from calf serum in the medium. This is supported by l) the

band being present in the same concentration in the control

medium, 2) the band being removed by passing it through

-35-

Sephadex G-200 and concentration by centrifugation, and 3)

the ultraviolet absorbance Spectrum. The 1.26 Specific gravity
band in CsCl gradients may be cell fragments or non-infective
virus. IBV causes granulation of cells and floating debris
extracellularly. This 1.26 Specific gravity material is not
dense enough to be pure protein. The sediment could contain
ribosomes, RNA and DNA of cellular origin. The material of
about 1.1 Specific gravity may be viral antigens or soluble
proteins released from the cell (Tevethia, 1964).

By the centrifugation methods employed, it was not possi—
ble to selectively separate the populations of IBV producing
either large or small plaques. Two populations are suggested
by the 0-D relationship of virus propagated in embryonating
chicken eggs (Singh, 1960) and by the DEAE separation of the
small and large plaques in mixed populations (Tevethia, 196M).
Also, herpes simplex was separated into two p0pulations by
isopycnic density gradient centrifugation (Roizman and Roane,
1961). However, with IBV the small and large plaques were
usually present in most fractions without regard to density.
Also the plaque isolates from low and high levels in the zonal
centrifuge tube reproduced progeny of similar density. There»
fore, small and large plaques are probably not caused by differ~
ences of the virus, but perhaps by CEKC differences, inhibitors
(interferon), virus adsorption problems or other variables.

The cell associated virus preparation was different from
the extracellular virus preparations. It was more heterogeneous

with the specific gravity ranging from 1.15 to 1.21 in sucrose

-36..

gradients. Irregular forms of a size associated with virus
were present in electron micrographs. AS the virus matures
at the cell surface, the small protrusions from the cell mem~
brane may be broken off by sonic vibration with release of
the virus. When the cells are ruptured, mature virus within
the cell would also be released, Instead of finding more
homogeneous viral particles within the cell, the virus was
more heterogeneous. This may be due to various size clumps
of virus enclosed in cellular material.

The prOpertieS of IBV determined by this study are similar

to those of the myxovirus group.

SUMMARY

1. The maximum amount of extracellular infectious
bronchitis virus (IBV) from chicken embryo kidney cell culture
(CEKC) was at 1.24 specific gravity as determined by iSOpycnic
density gradient centrifugation in cesium chloride. Lesser
amounts of virus were present in the range of 1.22 to 1.27
specific gravity. This salt inactivated some of the viral
particles.

2. With sucrose density gradient centrifugation, the
maximum amount of extracellular virus was at 1.19 Specific
gravity, and lesser amounts from 1.18 to 1.20 specific gravity.
The reduction of viral infectivity by sucrose was negligible.
When the virus suSpenSion was clarified by Sephadex G-200 and
centrifugally concentrated, the recovery of virus was low.

3. Using a partition cell method of centrifugation, the
sedimentation constant of IBV was 3348. The virus is a
sphere of approximately 80 mu diameter based on calculations
from the 8 value and 100 mu from electron microscOpy.

h. The range of Specific gravity of the cell associated
virus was greater than for extracellular virus. Extracellular
virus was a concentric Sphere, but intracellular virus was

irregular in shape according to electron microscopy.

.3?-

LITERATURE CITED

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polyoma virus. III. Correlation with biological
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Akers, T. G. 1963. Some cytochemical studies of the multi-
plication of infectious bronchitis virus in chicken
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University, East Lansing, Michigan.

Beaudette, F. H. and C. B. Hudson. 1937. Cultivation of the
virus of infectious bronchitis. J. A. V. M. A. 20:51-58.

Berry, D. M., J. G. Cruickshank, H. P. Chu and B. J. H. Wells.
1964. The structure of infectious bronchitis virus.
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Black, P. H., E. M. Crawford and L. V. Crawford. l96h.
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Brakke, M. K. 1958. Estimation of sedimentation constants

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Brown, W. E., S. C. Schmittle and J. W. Foster. 1962. A
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Buthala, D. H. 1956. Some properties of the avian bronchitis
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Corbo, L. J. and C. H. Cunningham. 1959. Hemagglutination
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Crawford, L. V. 1960. A study of the Hous sarcoma virus by
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Crawford, L. V. and E. M. Crawford. 1963. A comparative

study of polyoma and papilloma viruses. Virology 21:
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Cunningham, C. H. 1960. Recent studies on the virus of
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Cunningham, C. H. 1963. Laboratory guide in virology.

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-39-

Cunningham, C. H. and M. P. Spring. 1965. Some studies of
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Daniels, E., J. H. Mathews, J. W. Williams, P. Bender and
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Delaplane, J. P. and H. 0. Stuart. 1941. The modification
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Glasstone, S. and D. Lewis. 1960. Elements of physical chemi-
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Martin, B. G. and B. N. Ames. 1961. A.method for determining
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-ho-

Meselson, M., F. W. Stahl and J. Vinograd. 1957. Equilibrium
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Mohanty, B. S. and S. C. Chang. 1963. Development and ether
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Nazerian, K. 1960. Electron microscopy studies of the virus
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Pfefferkorn, E. B. and H. S. Hunter. 1963. Purification and
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-41-

Tanford, C. 1961. Physical chemistry of macromolecules.
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