H i; w .;~ w
H I I“? W ’3 ‘h w

L I} {i‘
\

 

BfiMUE‘ifiFLGOR’i-ZSCE 4E {3F AVIAN

éféFESTiC‘US ERMCHEHS VERUS AND

PfiEWCfiSTLE DESE‘XSE W333 5N
SENSLY 3ND QUALLY WFECTED
CELL CULWRES

Thesis for the Degzee of M. S.
WCHEGAN STATE UNIVERSETY
EUHN LOFTON BROWN
1969

THE-S‘s

 

 

 
  

 
  
  

1’ BINSING av -
HUN; 8: SBNS'
BUUK BINDERY INC.

LIBRARY B|NDERS
”BIKINI“. IICHIBMI .

    
   

ABSTRAC T

IMMUNOFLUORESCENCE OF
AVIAN INFECTIOUS BRONCHITIS VIRUS AND
NEWCASTLE DISEASE VIRUS IN SINGLY AND
DUALLY INFECTED CELL CULTURES
By

John Lofton Brown

On the basis of fluorescence microscopy, avian infectious
bronchitis virus (IBV) and Newcastle disease virus (NDV) were
specifically and differentially identified in single and dual infections
of primary chicken embryo kidney cells (CEKC). In singly infected
CEKC cultures, the size and number of foci of viral infected cells
was directly related to the viral concentration of the inoculum and
the time of viral replication. Evidence of interference of NDV by
IBV was observed in dual infections of CEKC. Replication of NDV
occurred equally well in baby hamster kidney cells (BHK-21) as in

CEKC, but there was no evidence that IBV infected BHK-21 cells.

IMMUNOFLUORESCENCE OF
AVIAN INFECTIOUS BRONCHITIS VIRUS AND
NEWCASTLE DISEASE VIRUS IN SINGLY AND

DUALLY INFECTED CELL CULTURES

By

John Lofton Brown

A THE SIS

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

MASTER OF SCIENCE

Department of Microbiology and Public Health

1969

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. Charles H.
Cunningham, Professor of Microbiology and Public Health, whoSe
guidance and encouragement throughout the investigation, and assis-
tance with manuscript preparation, not only contributed immeasurably
to the success of this study, but alsomade it a memorable experi-
ence.

I also wish to extend special thanks to Mrs. Martha P.
Spring, Department of Microbiology and Public Health, for her
patient assistance and invaluable instruction in proper cell culture
techniques.

In addition, appreciation is expressed to the Surgeon
General of the United States Army and the U. S. Army Veterinary
Corps who made possible my study at Michigan State University.

This study was supported in part by a grant-in-aid from

the United States Department of Agriculture.

ii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 3
MATERIALS AND METHODS . . . . . . . . . . . . . . . 7
Viruses . . . . . . . . . . . . . . . . . . . . . . . 7
Cell Cultures . . . . . . . . . . . . 7
Inoculation of Cells in Leighton Tubes . . . . . . . . . 10
Antisera. . . . . . . . . . . . . . . . . . . 10
Conjugation of Antiserum . . . . . . 11
Fluorescent Antibody Staining of Coverslip Cultures . . . 13
Microscopy . . . . . . . . . . . . . . . . . . . '. . 13
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 14
Relationship Between Concentration of Virus
and Time of Development of CPE . . . . . . . . . 14
Fluorescence of IBV as the Single Inoculum
in CEKC . . . . . . . . . 17
Fluorescence of NDV as the Single Inoculum
in CEKC . . . . . . . . . 18
Fluorescence of IBV and NDV as Dual Inoculum
in CEKC . . . . . . . . 18
Fluorescence of IBV and NDV as Single Inoculum
in BHK- 21 Cells . . . . . . . . . . . . . . . . . 20
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 32

LITERATURECITED...................35

iii

LIST OF TABLES

Table Page

1. Development of CPE in CEKC infected with
various dilutions ofIBV. . . . . . . . . . . . 15

2. Development of CPE in CEKC infected with
various dilutions of NDV . . . . . . . . . . . 16

3. Development of fluorescence and CPE in CEKC
infected with various PFU of IBV and NDV,

singly and in combinations . . . . . . . . . . 19

4. Development of CPE in BHK-Zl infected with
various dilutions of NDV . . . . . . . . . . . 21

iv

Table

LIST OF TABLES

Development of CPE in CEKC infected with
various dilutions of IBV .

Development of CPE in CEKC infected with
various dilutions of NDV

Development of fluorescence and CPE in CEKC
infected with various PFU of IBV and NDV,

singly and in combinations

Development of CPE in BHK-21 infected with
various dilutions of NDV

iv

Page

15

16

19

21

Figure

LIST OF FIGURES

Chicken embryo kidney cells 12 hours after
infection with 250 PFU of IBV. Two
isolated cells (arrows) are infected with
IBV. X500.

Chicken embryo kidney cells 24 hours after
infection with 250 PFU of IBV. X500.

Chicken embryo kidney cells 72 hours after
infection with 16 PFU of IBV. X500.

Chicken embryo kidney cells 60 hours after
infection with 100 PFU of IBV. The
large cytoplasmic fragments (arrows)
were characteristic of the CPE pro-
duced by IBV. X500.

Chicken embryo kidney cells 72 hours after
infection with 100 PFU of IBV. X1000.

Chicken embryo kidney cells approximately
12 hours after infection with 250 PFU
of IBV. Note the antigen is distributed
both diffusely and within spheroid
granules. X 1000. . .

Small syncytium in CEKC 24 hours after
infection with 125 PFU of IBV. X1000.

Chicken embryo kidney cells 12 hours after
infection with 10 PFU of NDV. Small
spherical foci of early NDV replica-
tion (arrows). X500. . . .

Page

23

23

24

24

25

25

26

26

Figure

10.

11.

12.

13.

14.

15.

16.

17.

Page

Chicken embryo kidney cells 24 hours after
infection with 70 PFU of NDV. The
yellow autofluorescence is apparently
not viral related as it is also present in
non-infected controls. X500. . . . . . . . . . 27

Chicken embryo kidney cells 36 hours after
infection with 70 PFU of NDV. X500. . . . . . 27

Early stages of replication of NDV in CEKC 36
hours after infection with 35 PFU of NDV.
X500. . . . . . . . . . . . . . . . . . . . 28

Terminal stages of NDV replication in CEKC
24 hours after infection with 140 PFU of
NDV. X1000. . . . . . . . . . . . . . . . 28

At 24 hours after infection with IBV 100:
NDV 10, NDV-specific fluorescent anti-
body indicates NDV within small clusters
of cells and in cells radiating from the
clusters.X500...............29

At 36 hours after infection with IBV 100:
NDV 10, NDV-specific fluorescent anti-
body indicates interference in replication
of NDV. Infection has failed to spread
from the clusters observed at 24 hours.
X500..............,......29

At 48 hours after infection with IBV 100:
NDV 10, NDV-specific antibody indicates
replication of NDV has been limited to a
small segment of the cell population.
X500....................30

At 48 hours following infection with IBV 100:
NDV 10, IBV-specific antibody indicates
most cells are infected with IBV. X500. . . . 30

Baby hamster kidney cells 8 hours after
infection with 560 PFU of NDV. X500. . . . . 31

vi

INTRODUC TION

Study of viruses using a living indicator system requires
cognizance of the host cell' 8 susceptibility to a virion and recog-
nition of any interrelationship between different viral agents present
in the host cell population. Infectious bronchitis virus (IBV) and
Newcastle disease virus (NDV) are of particular interest in this
regard because the chicken is a natural host for both viruses and
there is a high probability of their coexistence under both laboratory
and environmental conditions.

The capacity of IBV to exert interference upon NDV repli-
cation has been reported and has even been used as a means of
assaying IBV (6). However, the interference phenomenon could
mask the detection of a low concentration of NDV in the presence of
a high concentration of IBV. Embryonating chicken eggs and avian
cell cultures are commonly used for growth of IBV and NDV. To
assure the complete purity of viral stocks, it is essential to know if
small amounts of NDV or IBV can be detected in the presence of the

other virus and their reciprocal influence.

Using the fluorescent antibody technique, this study was
designed to demonstrate if low levels of each virus could be detected
in the presence of large numbers of the other, the time of viral
development, the cytopathic effect, and possible differences in the

fluorescence of the two viruses during synthesis within the cell.

LI TERA TURE REVIEW

Avian infectious bronchitis. virus (IBV) has an average
diameter of 80 to 100 mil. with distinctive surface projections 20 mp,
long, club-shaped in outline, and attached to the viral surface by a
narrow stalk (11, 12). It has an ether sensitive lipoprotein coat
(29) and a ribonucleic acid core (2). It is presently not classified
in any recognized major viral group (44), but recognition by Almeida
and Tyrrell in 1967 (3) of the similarity between IBV and recently
isolated human respiratory viruses, B814 and 229E, indicated the
existence of a previously unrecognized viral group. Additional work
has confirmed these findings (7, 28, 31), and it was also pointed out
that mouse hepatitis virus (MHV) possessed similar properties.
This has prompted a group of virologists to propose that IBV,
several human respiratory viruses, and MHV are members of a
unique viral group (4). Because their electron microscopic appear-
ance resembles the solar corona, they have suggested that this

group be called the coronaviruses, with IBV as the prototype.

Infectious bronchitis virus can be propagated readily in

embryonating chicken eggs, the most usual route being the allantoic

cavity (16). Propagation in cell culture is possible with several
primary avian cell types (17), but one of the most sensitive and
frequently used is the primary chick embryo cell (CEKC) (27).
Infection begins when viral particles adsorb to the outer membrane
of the CEKC. There is a 4 hour eclipse period. Maximal viral
yield is reached in 48 hours, maintained through 72 hours, and
then gradually declines (2, 17).

Newcastle disease virus (NDV), a member of the
paramyxovirus group (44), is an RNA- containing, enveloped,
roughly spherical pleomorphic particle, with a diameter of approx-
imately 100-200 ml], (19, 22). The helical nucleocapsid is enclosed
by an ether sensitive lipopritein coat with surface spikes about 8mg,
in length and spaced about 8—10 m/J, apart (42).

Newcastle disease virus multiplies to high titer when inocu-
lated into the embryonating chicken egg (25) and grows in a wide
variety of cells of avian and mammalian origin (24). In chicken
embryonic lung cell cultures, NDV has a latent period of 2 hours
and a period of viral release of 4 hours, with approximately 50
plaque-forming units released from each cell (20).

Separation and characterization of virus-Specific antigens
is reported for both IBV and NDV. Three antigenically distinct

soluble antigens are associated with IBV and can be differentiated

on the basis of thermostability, size, buoyant density, and
sensitivity to enzymes (40). Newcastle disease virus possesses
two distinct and separable type-specific antigens, a surface lipo-
protein antigen (hemagglutinin or V-antigen) and an internal or
ribonucleOprotein antigen (nucleocapsid or S-antigen) (38). The
antigens can be differentiated on the basis of enzyme activity,
hemagglutinating, complement-fixing, and enzyme sensitivity.

The earliest report of detection of IBV by fluorescence
microscopy indicated the presence of intranuclear IBV antigen (2 9).
Later work by Lukert has contradicted these findings (27). Using
IBV-infected chicken embryo kidney, liver, lung, and fibroblast
cell cultures, no nuclear fluorescence was detected. Development
of IBV antigen was essentially the same in all cell types. The first
fluorescence occurred 4 hours after infection, primarily in the
nuclear region, and progressed peripherally until the cytoplasm
contained diffuse and granular fluorescence. Syncytia developed
within 7 to 8 hours and reached their maximum size at 18 to 20
hours.

Fluorescence microscopy has been used to study cellular
multiplication of NDV (14, 35, 37, 43, 45). Using specific antibody
against S- and V-antigens by the direct test, Reda, Rott, and

Schafer (35) reported that distinct fluorescent foci appeared around

the nucleus in about 3 hours. These foci increased in size and
brightness with time and occurred throughout the cytoplasm. The
V-antigen was detectable shortly after the S-antigen and at about
the 7th hour it was distributed throughout the cytoplasm.

Viral interference occurs when two viruses or two strains
of a single virus infect a single cell, and one virus interferes with
the growth or replication of the other (36). Interference of IBV with
NDV has been demonstrated in chickens (21), embryonating chicken
eggs (33), and chick embryo kidney cells (7). Regardless of the test
system used, interference was dependent upon an excess of IBV.
Interference apparently occurs intracellularly because when NDV
was inoculated into developing chicken embryos 4 hours prior to the
administration of IBV, interference was still demonstrated. The
interference mechanism does not appear to involve blocking of

cellular receptor sites or to be influenced by interferon.

MATERIALS AND ME THODS

Viruses

The Beaudette strain of avian infectious bronchitis virus
(IBV-42) adapted to chick embryo kidney cells (CEKC) (18) was
used and assayed in primary CEKC cultures as plaque-forming units
(PFU) (16). Primary CEKC in petri dishes were inoculated with
2. 2 X 105 PFU of the 130th passage of IBV. At 48 hours the extra-
cellular fluid was harvested, pooled, and distributed in 1 ml quan-
tities into glass vials for storage at -90 C. The virus contained

2. 5 X 107 PFU per m1.

Newcastle disease virus (NDV), Texas-GB strain, 1. 2 X 106
PFU in 0. 1 ml, was used to inoculate 10-day-old embryonating
chicken eggs via the allantoic cavity. All embryos died within 28
hours and were chilled at 4 C for 6 hours prior to collection of
allantoic fluid. The fluid was dispensed in 1 ml amounts into glass
vials and stored at -60 C. The titer of the virus was 2. 8 X 109 PFU

per ml assayed in CEKC (16).

Cell Cultures

 

Primary CEKC prepared from 17-day-old chicken embryos

(16) were suspended in growth medium consisting of Medium 199 with

2 mM L—glutamine, Grand Island Biological Company (GIBCO),
fortified with amino acids and vitamins of Eagle' s basal minimum
essential medium (GIBCO), 0. 1% sodium bicarbonate, 100 units/ml
penicillin, 100 [.Lg/ml dihydrostreptomycin, and 50 units/ml
Mycostatin (Squibb). Newborn calf serum, 5% final concentration,
was then added. For plaque assay, a 1:100 suspension of packed
cells (approximately 107 cells/ml) was dispensed in 4 ml quantities
into 15 X 60 mm plastic petri dishes (Falcon Plastics, Los Angeles,
Calif. ). Leighton tubes containing a coverslip (9 X 22 mm) were
seeded with 1 ml of a 1:400 suspension (approximately 2. 5 X 106
cells/ml) of packed cells. All cell cultures were incubated in an

atmosphere of 8% CO and 80—85% relative humidity at 37 C. Mono-

2
layers formed in the Leighton tubes within 24 hours and in the petri
dishes within 48 to 72 hours.

Baby hamster kidney cells (BHK—21) (Microbiological Asso-
ciates, Bethesda, Md. ) were also used starting with the 150th pas-
sage. The cells were propagated in milk dilution bottles with
screwcaps as a closed system. The growth medium was Eagle' 3
minimum essential medium with Hanks' solution (HMEM) and 2 mM
L-glutamine, without sodium bicarbonate (GIBCO), 50 mcg/ ml

aureomycin, 100 units/ml tylosin tartrate, and adjusted to pH 7. 4

with 7. 5% sodium bicarbonate.

After development of a confluent monolayer in 3 to 4 days,
the cells were rinsed twice with 5 ml of phosphate buffered saline
(PBS) without Ca++ or Mg++ (39). The PBS was decanted and
approximately 4 ml of buffered 0.25% trypsin containing 6. 8 g/L
sodium chloride, 0.4 g/L potassium chloride, 1 g/L glucose,

2. 2 g/L sodium bicarbonate, and 0.005 g/L phenol red (GIBCO
Solution A) at room temperature was added to the cells. In approxi-
mately 2 minutes, the trypsin solution was decanted and the bottle
was inverted for another 2 minutes or until the cells detached from
the surface. The cells were then suspended in growth medium at a
volume 3 times that of the original medium and agitated vigorously.
Fetal calf serum was added to a final concentration of 10%.

The suspension of cells was dispensed in 15 ml amounts
into bottles and incubated at 37 C. The medium was replaced at
approximately 2 day intervals within 7 days with equal parts of the
growth medium and nutrient mixture F10 with 2 mM L-glutamine
(GIBCO), 100 units/ml penicillin, 100 [.Lg/ml dihydrostreptomycin,
and 0. 0075% sodium bicarbonate. There was a final concentration
of 5% fetal calf serum.

Leighton tubes were seeded with 1 ml of trypsinized BHK-
21 resuspended in growth medium at the original volume. Mono-

layers were formed within 24 hours.

lO

Inoculation of Cells in Leighton Tubes

 

The extracellular fluid from CEKC was decanted and the
. . ++ ++ . .
cells were washed With PBS WIthout Ca or Mg . Dilutions of the
viruses to the PFU desired were made at approximately 0 C in PBS
. ++ ++ . .

w1thout Ca or Mg but contaimng 2% newborn calf serum. An
inoculum of 0.2 ml was pipetted onto each coverslip culture. The
cultures were reincubated for 60 to 90 minutes and then 2 ml of
equal parts Medium 199 and F10 containing 2% newborn calf serum
was added.

Tubes of BHK-21 were handled similarly except that fetal
calf serum was substituted for the newborn calf serum in the
diluent. In the maintenance medium HMEM replaced Medium 199.

The cells were first examined by bright field microscopy
at 12 hour intervals for cytopathogenic effects. The coverslips
were then removed for staining and examination by fluorescent

antibody mi cros copy.

Antisera

The IBV antisera from specific pathogen free chickens
was kindly supplied by Dr. D. D. Oshel, Agricultural Research
Service, Ames, Iowa. The neutralization index against IBV-42 was
6. 3 X 105 as determined by the embryo lethal dose50 (ELDSO) in

chicken embryos.

11

Dr. M. S. Hofstad, Iowa State University, Ames, Iowa,
kindly supplied NDV-antiseria from specific pathogen free turkeys.

The neutralization index ELD was greater than 5. 6 X 106 against

50
Texas-GB and less than 101 against IBV-42.

Conjugation of Antiserum

 

The antisera were conjugated according to a method
described by Lukert (27). Serum was fractionated by the dropwise
addition of one volume of saturated ammonium sulfate into two
volumes of serum with constant mixing in an ice bath. Precipitated
globulins were centrifuged at 2000 g. for 1.5 minutes and redissolved
in 0. 85% NaCl solution equal to the original volume of serum. This
procedure was repeated twice. The final precipitate was restored
to one -half the original volume and dialyzed at 4 C against at least
100 volumes of 0.85% NaCl. The saline solution was changed 3
times within 24 hours or until it was free of SO4++. Any precipitate
in the immunoglobulin fraction at the last dialysis was removed by
centri fugati on.

The concentration of protein in the immunoglobulin solution

was determined (26) using crystalline bovine serum albumin as the

standard. The solution was then diluted to contain 10 mg protein

per ml of 0.85% NaCl.

12

A volume of 0. 5 M sodium carbonate-sodium bicarbonate
buffer solution, pH 9.0, equal to 1/10 the immunoglobulin volume
was used to dissolve crystalline fluorescein isothiocyanate (FITC)
(Baltimore Biological Laboratory, Cockeysville, Md.) at the rate
of 25 Mg FITC per mg of protein in the immunoglobulin solution.
The FITC-carbonate buffer solution was then added to the immuno-
globulin solution and stirred at 4 C for 16 to 20 hours. The mixture
was then added to a column of Sephadex G-25, coarse (Pharmacia
Fine Chemicals, Uppsala, Sweden) in optimum amounts for maxi-
mum differential elution of the FITC labeled globulin from free FITC
and eluted with PBS. The column of Sephadex was prepared with
0.01 M PBS, pH 7. 2, and had a height-to-diameter ratio or 15:1.

The elute was added to a PBS—rabbit liver powder (RLP)
(Baltimore Biological Laboratory) slurry (2. 5 ml : 1 mg) to give a
final ratio of 1 mg of protein in the immunoglobulin. solution to
20 mg of RLP in the slurry. This mixture was stirred constantly
16 to 20 hours at 4 C. RLP was sedimented by centrifugation at
2000 g. for 30 minutes followed by 45, 000 g. for 90 minutes at 4 C.
The supernatant fluid was passed througha filter (Millipore Filter
Corp. , Bedford, Mass. ), pore size 0.30 microns, and stored in
glass vials at -20 C. Just prior to use, the conjugate was thawed,
diluted 1:4 with PBS, pH 7. 2, and centrifuged at 2000 g. for 15

minutes to remove any precipitate.

13

Fluorescent Antibody Staining
of Coverslip Cultures

 

 

Staining was also by the method of Lukert (27). Appropriate
coverslip cultures from the Leighton tubes were washed in PBS,
pH 7. 2. While wet, they were momentarily immersed in acetone
for partial dehydration. They were then fixed in another container
of acetone for 3 to 5 minutes at room temperature and air dried.
A few drops of labeled antibody were placed on the coverslips which
were incubated for 45 minutes in a moist chamber at 37 C. The
coverslips were immersed in tap water at room temperature for
3 minutes with gentle mixing, after which they were air dried. A
drop of equal volumes glycerin and PBS, pH 7. 2, was used for

mounting the coverslips on a slide.

Microscopy

 

Cells were examined with an AO-Spencer ”Microstar”
micros00pe illuminated with an Osram HBO-200 mercury vapor arc
lamp. The excitation filter was the Schott BG-12 and the barrier
filter the Schott OG-l. Photomicrographs were made with high
speed Ektachrome daylight film using exposure times of 2 to 5

minutes.

RESULTS

Relationship Between Concentration of Virus
and Time of Development of CPE

 

 

Infectious bronchitis virus

 

There was direct relationship between the number of PFU
of IBV in the inoculum and the time when cytopathic effects (CPE)
were present (Table 1). With 250 PFU, CPE was observed as early
as 36 hours, but with the more dilute inoculum, -1. 8 log units, CPE
was not evident in some cultures until as late as 84 hours. There
was no CPE in dilutions greater than -1. 8 log units indicating that
infectious virus was not present in the inoculum. Characteristic
cell alterations were the loss of cytoplasmic detail, formation of
circumscribed cellular aggregates and syncytia, followed by lysis

of cells and their detachment from the surface of the tube.

Newcastle disease virus

 

The CPE of cultures inoculated with 280 PFU of NDV and
with dilutions as high as -1. 8 was first detected at 36 hours (Table 2).

However, with -2. 1 to -3. 0 dilutions of virus the time of development

14

15

Table 1. -— Development of CPE in CEKC infected with various
dilutions of IBV.

 

 

Log dilution of
250 PFU IBV
inoculated onto
each of 4
Leighton tube
cultures

Hour post-inoculation

 

12

24

36 48 6O 72 84

 

 

 

 

+ +++

++++

++++

++

 

 

 

 

 

+ = number of cell cultures with CPE.

Table 2. -- Development of CPE in CEKC infected with various

dilutions of NDV.

16

 

 

Log dilution
of 280 PFU
NDV inocu-
lated onto
each of 4
Leighton tube
cuhures

Hour post-inoculation

 

12

24

36

48

60

72

84

96

108

 

 

 

 

++++

++++

++++

++++

++++

++++

++++

 

 

 

 

 

 

 

+ = number of cell cultures with CPE.

17

of CPE ranged from 36 to 108 hours. At dilutions greater than

—3. 0, CPE did not deve10p, indicating that infectious virus was not
present in the inoculum. With the exception of a more extensive
lysis of the monolayer by NDV, the CPE produced by NDV was
similar to that produced by IBV.

Fluorescence of IBV as the Single Inoculum
in CEKC

 

At 12 hours fluorescence was present in widely separated
individual cells or in 2 or 3 adjoining cells in cultures inoculated
with 125 or 250 PFU (Figure 1). By 24 hours, fluorescence was
also evident in cultures infected with 63 PFU (Figure 2). Both the
size and number of fluorescent foci increased with time, indicating
the rate of replication and release of new viral infectious units was
directly dependent upon the concentration of virus in the inoculum
(Figures 3, 4, 5).

During replication of the virus, the first evidence of
cellular infection was the development of a diffuse, perinuclear
antigen which gradually spread peripherally with subsequent devel-
opment of small, spheroid packets throughout the cytoplasm

(Figures 6 and 7). Fluorescence was not observed in the nucleus.

18

Fluorescence of NDV as the Single Inoculum
in CEKC

 

Between 12 and 24 hours after infection, the size and
number of fluorescent foci increased and were directly related to
the viral concentration of the inoculum and the length of time of
viral replication (Figures 8 and 9). With all NDV infected cultures,
the inevitable outcome was the development of fluorescence within
essentially 100% of the cells comprising the monolayer. There was
an ultimate total disintegration and lysis of infected cells (Figure 10).

The earliest evidence of NDV replication was small, bril-
liantly fluorescing, segregated cytoplasmic bodies near the nucleus.
These bodies subsequently developed into large polymorphous forms
(Figures 11 and 12). Shortly following the first appearance of the
segregated bodies there was development of a diffuse fluorescence
throughout the cytoplasm.

Fluorescence of IBV and NDV as Dual Inoculum
in CEKC

 

When cultures were dually infected with PFU combinations
of IBV 10:NDV 10, 10:100, and 100:100, respectively, there was no
significant difference in the time of appearance of fluorescence as
compared to that when the cultures were singly infected (Table 3).

However, the time required for complete cell destruction differed

19

6.2530 :8 .Ho mMmOAUDZ

ll
=fl:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6.2530 Zoo Piano no“ .****nun-* u an
.8330 Zoo Pause no.“ .++++-u:+ .mocoomononfi mo bacon—E u +
*** **
mm 0» up
it I: ++++ ++++
**** *3; as." * om
I: it +++ ++++ i: I: it it I: ++++ ++++
33% is: “new * 3.3% *5" Leon *3" new A" we
++++ ++++ ++ ++++ ++++ +++ .T_.++ ++ 3;; ++++ +.I.+ ++++
in *1? an new * mm
++++ +++ ++ +++ ++++ ++ +++ + ++++ +++ +++ ++
++ + ++ + ++ ++ ++ ++ + em
+ + + + NH
>QZ >mH >QZ >m: >QZ >m: >QZ >mH >QZ >QZ >m: >m: c3335
OCH 00H S 03 o2 oH 3 A: 2: 2 ca 3 .pm .
am
musom
may; cam Dbnm mo ADQEflZ

 

 

 

.mcoflacwnaoo 5 Cam Ewan.” .>QZ

cam >m: mo Dbm msoflmew 3:3 Douommfi UMHU E mmU Dam moamommponfi mo unmamofioarmfl: .m 2an

20

from that when either virus was replicating in a singly infected
culture. In the mixture of IBV 10:NDV 100, NDV infected and lysed
cells throughout the culture by 60 hours, which was before IBV had
infected a number of cells comparable to its concentration. The
viruses at IBV 10:NDV 10 and 100:100 completely destroyed the

cells in 60 hours, but at IBV 100:NDV 10 the cells were not destroyed
until 72 hours.

Fluorescence in cells infected with IBV 100:NDV 10
indicated that IBV interfered with the replication of NDV. At 24
hours, NDV was apparent within several small clusters of cells and
in cells radiating from the clusters (Figure 13). At the same time,
IBV-induced sycytia were present. Following this, IBV was more
pronounced than NDV and at 48 hours IBV completely dominated the
cell culture except for scattered areas of NDV infected cells (Fig-
ures 14, 15, 16).

Fluorescence of IBV‘and NDV as Single Inoculum
in BHK-21 Cells

 

 

Infectious bronchitis virus, 5 X 105 PFU, did not infect

the cells based on the absence of CPE or fluorescence, up to and
including 60 hours after inoculation.
Newcastle disease virus replicated equally as well in

BHK-21 as in CEKC (Table 4). The relationship between the time

21

Table 4. -—Development of CPE in BHK-21 infected with various
dilutions of NDV.

 

 

 

 

Log dilution Of 250 Hour ost-inoculation
PFU NDV Inoculated p
onto each of 3
Lelghton tube 12 24 36 48 60 72
cultures
0 + ++
-1.0 +++
-2- 0 + ++
-3. 0 + ++
-4. 0 +
-5. 0

 

 

 

 

 

 

 

+ = number of cell cultures with CPE.

22

of deve10pment of CPE observed by bright field microscopy and the
manner of replication observed by fluorescence microscopy was

essentially the same as in CEKC (Figure 17).

23

 

Fig. 1. -- Chicken embryo kidney cells 12 hours after infection with
250 PFU of IBV. Two isolated cells (arrows) are infected
with IBV. X500.

 

L,-_.~_._ _.L.-_,_-_ ____ L_. ,,-____ L

 

Fig. 2. -- Chicken embryo kidney cells 24 hours after infection with
250 PFU of IBV. X500.

24

 

Fig. 3. -- Chicken embryo kidney cells 72 hours after infection with
16 PFU of IBV. X500.

 

 

Fig. 4. -- Chicken embryo kidney ‘cells 60 hours after infection with
100 PFU of IBV. The large cytoplasmic fragments (arrows)
were characteristic of the CPE produced by IBV. X500.

 

25

   

Fig. 5. -- Chicken embryo kidney cells 72 hours after infection with
100 PFU of IBV. X1000.

~- _~*_

 

, D
L'— " _ * ..,-.-, V” —v—- _‘~,__(__A —' , " , ~--—-4

 

Fig. 6. -- Chicken embryo kidney cells approximately 12 hours after
infection with 250 PFU of IBV. Note the antigen is dis-
tributed both diffusely and within spheroid granules. X 1000.

26

 

Fig. 7. -- Small syncytium in CEKC 24 hours after infection with
125 PFU of IBV. X1000.

  

L

Fig. 8. -- Chicken embryo kidney cells 12 hours after infection with
10 PFU of NDV. Small sphericalfoci of early NDV repli-
cation (arrows). X500.

 

 

 

27

 

Fig. 9. -- Chicken embryo kidney cells 24 hours after infection with
70 PFU of NDV. The yellow autofluorescence is apparently
not viral related as it is also present in non-infected con-
trols. X500.

 

 

Fig. 10. -- Chicken embryo kidney cells 36 hours after infection with
70 PFU of NDV. X500.

 

28

 

L‘ ‘ ' ' 7‘ "'—' ' ‘A’ ' ‘7‘

Fig. 11. -- Early stages of replication of NDV in CEKC 36 hours
after infection with 35 PFU of NDV. X500.

 

L. __ ,1. _- ._ - _-,__._ _____-_,

Fig. 12. -- Terminal stages of NDV replication in CEKC 24 hours
after infection with 140 PFU of NDV. X 1000.

29

 

Fig. 13. --At 24 hours after infection with IBV 100:NDV 10, NDV-
specific fluorescent antibody indicates NDV within small

clusters of cells and in cells radiating from the clusters.
X500.

 

 

Fig. 14. --At 36 hours after infection with IBV 100:NDV 10, NDV-
specific fluorescent antibody indicates interference in
replication of NDV. Infection has failed to spread from
the clusters observed at 24 hours. X500.

30

 

Fig. 15. —-At 48 hours after infection with IBV 100:NDV 10, NDV-
specific antibody indicates replication of NDV has been
limited to a small segment of the cell population. X500.

— , .—

 

—' , - ‘7 7 ' —— * - ~' ' 7 if v * —*‘A-A* a

Fig. 16. --At 48 hours following infection with IBV 100:NDV 10, IBV-
specific antibody indicates most cells are infected with
IBV. X500.

31

   

 

t. _-_-__-,- in" - - i.

 

Fig. 17. -- Baby hamster kidney cells 8 hours after infection with
560 PFU of NDV. X500.

DISCUSSION

According to electron microscopy, morphogenesis of IBV
in the chorioallantoic membrane of chicken embryos (9) and in
chicken embryo fibroblasts (31) appeared to occurwithin the cyto-
plasm by budding of viral structures into cytoplasmic vesicles or
cisternae. Avian cells infected with IBV were demonstrated by
fluorescence microsc0py to contain diffuse and granular intra—
cellular antigen (27). When stained with fluorochromes (2, 10),
the fluorescence was from IBV ribonucleoprotein developing as
intracytoplasmic granules. The intracytoplasmic fluorescence
observed in the present study further supports these reports.

Using electron, phase, and bright field microscopy (1, 13,
15), intracytoplasmic inclusion bodies were detected in cells
infected with NDV. Other reports of multiplication of NDV, studied
by fluorescence microscopy (14, 32, 35, 41, 43, 45) describe
intracytoplasmic fluorescence similar to that observed in the
present study. Reda, Rott, and Schafer (35) determined that the
nucleoprotein or S-antigen was localized within the segregated

fluorescent cytoplasmic bodies, and the hemagglutinin or V-antigen

32

33

was diffusely located within the cytoplasm. The large masses of
S-antigen appeared as inclusion bodies when examined by phase
microscopy. In the present study, inclusion bodies were the most
characteristic fluorescence feature of NDV replication.
On the basis of the time of appearance of CPE in CEKC, ”B
the replication cycle of NDV is much shorter than that of IBV and ‘1

lesser amounts of NDV can be detected. I

 

 

Neutralizing antibody was used in this study and the ’ “‘1
fluorescence in infected cells was considered to be related to a viral
V-antigen-antibody complex, although S-antigens might have been
present.

It was clearly evident that IBV and NDV could be specifi-
cally and differentially identified by fluorescence microscopy in
single and dual infections of the same culture. There was no non—
specific fluorescence.

Of particular importance was the evidence of interference
of NDV by IBV when the ratios were 100:10 PFU, respectively.

This confirms previous reports of interference of NDV by IBV using
chickens, chicken embryos, and cell cultures as the indicator host
(5, 7, 8, 21, 33). So far as known, this has not been reported on
the basis of fluorescence microscopy. Normally, NDV alone would

haveeinfected all cells, but instead, IBV was dominant. Another

34

manifestation attributable to IBV interference was that destruction
of cells in dually infected cultures occurred later than in singly
. infected NDV cultures of equal viral concentration.

The precise mechanism of IBV interference is not under-
stood, but most work implies intracellular interference with no
demonstrable involvement of interferon (33, 34). Intracellular
interference is also indicated in this study. The invasiveness of
NDV and rapid replication of infectious virions was greater than
that of IBV; therefore, it does not seem feasible that the more
slowly replicating IBV could dominate a cell culture by simply
blocking viral cellular receptor sites. In infections in which there
was delayed cellular destruction, NDV had initially infected the
cells, but IBV altered the further synthesis of NDV.

The results obtained with BHK-21 were similar to those
reported using African green monkey kidney cells (23), in that
BHK-21 supported replication of NDV, but not IBV. Therefore,
BHK—21 provides another means of differentiating IBV and NDV
using a non-avian system.

The diffuse and granular fluorescence associated with IBV
infection suggests a similarity to the fluorescence of S- and V-
antigens within cells infected by NDV. Further studies using puri—

fied S- and V-antigens of IBV should provide an answer.

 

LI TE RA TURE CI TED

 

 

LI TERATURE CI TED

. Adams, R. W., and A. M. Prince. 1957. An electron micro—
scope study of incomplete virus formation. J our. Exptl.
Med. 106: 617—626.

. Akers, T. G., and C. H. Cunningham. 1968. Replication and
cyt0pathogenicity of avian infectious bronchitis virus in
chicken embryo kidney cells. Arch. Ges. Virusforsch.
_2_5: 30-37.

. Almeida, J. D., and D. A. J. Tyrrell. 1967. The morphology
of three previously uncharacterized human respiratory
viruses that grow in organ culture. J. Gen. Virol. 1:
175-178. ’

. Almeida, J. D., D. M. Berry, C. H. Cunningham, D. Hamre,
M. S. Hofstad, L. Mallucci, K. McIntosh, and D. A. J.
Tyrrell. 1968. Coronaviruses. Nature 220: 650.

. Bankowski, R. A., R. W. Hill, and L. G. Raggi. 1957.
Response of eight—week-old susceptible chickens to New-
castle disease (B-l) andAnfectious bronchitis viruses.
Avian Dis. 1: 195-207.

. Beard, C. W. 1968. An interference type of serological test
for infectious bronchitis virus using Newcastle disease
virus. Avian Dis. 1_2: 658-665.

. Beard, C. W. 1967. . Infectious bronchitis virus interference

with Newcastle disease virus in monolayers of chicken
kidney cells. Avian Dis. _1_1: 399-406.

. Beaudette, F. R. 1950. Infectious bronchitis and Newcastle
disease. Canad. J. Comp. Med. _1_5: 65-71.

35

“-1 K _-

 

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

36

. Becker, W. B., K. McIntosh, J. H. Bees, and R. M. Chanock.

1967. Morphogenesis of avian infectious bronchitis virus
and a related human virus (strain 229E). Jour. Virol. 1:
1019-1027.

Berry, D. M. 1967. Intracellular development of infectious
bronchitis virus. Nature 216: 293-294.

Berry, D. M., and J. D. Almeida. 1968. The morphological
and biological effects of various antisera on avian infectious
bronchitis virus. J. Gen. Virol. _3: 97-102.

Berry, D. M., J. G. Cruickshank, H. P. Chu, and R. J. H.
Wells. 1964. The structure of infectious bronchitis virus.
Virology a: 403-407.

Brandt, C. D. 1958. . Inclusion body formation with Newcastle
disease and mumps viruses in cultures of chick embryo
cells. Virology _5_: 177-191.

Brandt, C. D. 1961. Cytopathic action of myxovirus on cultured
mammalian cells. Virology 14: 1-10.

Butler, M. P. 1965. Avian viruses in adult cells in vitro. A
comparison of fowlpox, bronchitis, laryngotracheitis, and
Newcastle disease viruses in tissue cultures of embryonic
and adult fowl cells. Virology 2_5_: 454-468.

Cunningham, C. H. 1966. A laboratory guide in virology, 6th
ed. Burgess Publishing Co. , Minneapolis.

Cunningham, C. H. 1960. Recent studies on the virus of
infectious bronchitis. Am. J. Vet. Res. 2_1: 498-503.

Cunningham, C. H. , and M. P. Spring. 1965. Some studies
of infectious bronchitis virus in cell culture. Avian Dis.
2: 182-193.

Dawson, D. M., and W. J. Elford. 1949. The investigation
of influenza and related viruses in the electron microscope
by a new technique. J. Gen. Microbiol. 3: 298-301.

Franklin, R. M., H. Rubin, and C. A. Davis. 1957. The pro-
duction, purification, and properties of Newcastle disease
virus labeled with radiophosphorus. Virology 3: 96-114.

 

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

37

Hanson, L. E., F. H. White, and J. O. Alberts. 1956. Inter-
ference between Newcastle disease and infectious bronchitis
viruses. Am. J. Vet. Res. 1'_7_: 294-298.

Horne, R. W., and A. P. Waterson. 1960. Ahelical structure
in mumps, Newcastle disease and Sendai viruses. J. Mol.
Biol. _2_: 75-77.

Lacey, R. B. 1968. The use of serially propagated African
green monkey kidney cells in the detection of a latent New-
castle disease virus in chicken embryos. Ph. D. Thesis,
Michigan State University.

 

Lief, F. S. 1966. Myxoviruses, p. 246-312. EJ. E. Prier
(ed. ), Basic medical virology. Williams 8: Wilkins Co. ,
Baltimore.

 

Liu, C. , and F. B. Bang. 1953. An analysis of the difference
between a destructive and a vaccine strain of Newcastle
disease virus in the chick embryo. J. Immunol. E); 538-
548.

Lowry, O. H., N. J. Rosebrough, L. Farr, and R. J. Randall.
1951. Protein measurement with the folin phenol reagent.
J. Biol. Chem. 193: 265-275.

Lukert, P. D. 1966. Immunofluorescence of avian infectious
bronchitis virus in primary chicken embryo kidney, liver,
lung, and fibroblast cell cultures. Arch. Ges. Virusforsch.
19: 265-272.

McIntosh, K. L., J. H. Dees, W. B. Becker, A. Z. Kapikian,
and R. N. Chanock. 1967.. Recovery in tracheal organ

cultures of novel viruses from patients with respiratory
disease. Proc. Natl. Acad. Sci. U. S. 5'1: 933-940.

Mohanty, S. B., H. M. DeVolt, and J. E. Faber. 1964. A
fluorescent antibody study of infectious bronchitis virus.
Poultry Sci. 43: 179-182.

Mohanty, S. B., and S. C. Chang. 1963. Development and
ether sensitivity of infectious bronchitis virus of chickens
in cell cultures. Am. J. Vet. Res. 23: 822-826.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

38

Nazerian, K., and C. H. Cunningham. 1968. Morphogenesis
of avian infectious bronchitis virus in chicken embryo
fibroblasts. J. Gen. Virol. 3: 469-470.

Prince, A. M., and H. S. Ginsberg. 1957. Immunohistochemi-
cal studies on the interaction between Ehrlich ascities tumor
cells and Newcastle disease virus. J. Exptl. Med. 105:
177-188. _

Raggi, L. G. , and G. G. Lee. 1963. Infectious bronchitis
virus interference with the growth of Newcastle disease

virus. I. Study of interference in the chicken embryo.
Avian Dis. 1: 106-122.

Raggi, L. G. , and G. G. Lee. 1964. Infectious bronchitis
virus interference with growth of Newcastle disease virus.
11. Interference in chickens. Avian Dis. 8: 471-480.

Reda, I. M., R. Rott, and W.. Schafer. 1964. Fluorescent
antibody studies with Newcastle disease infected cell
systems. Virology 2_2_: 422-425.

Rhodes, A. J., and C. E. van Rooyan. 1962. Textbook of
virology, 4th ed. Williams and Wilkins Co. , Baltimore.

Rodriquez, J. E., and W. Henle. 1964. Studies on persistent
infections of tissue cultures. V. The initial infection
stages of L (MCN) cells by Newcastle disease virus. J.
Exptl. Med. i9: 895-922.

Rott, R. 1964. Antigenicity of Newcastle disease virus,
p. 133-146. 1_nR. P. Hanson (ed.), Newcastle disease
virus. An evolving pathogen. The University of Wiscon-
sin Press, Madison and Milwaukee.

Schmidt, N. J. 1964. Tissue culture methods and procedures
for diagnostic virology, p. 78-176. In E. H. Lenette and
J. N. Schmidt, Diagnostic procedure-s— for viral and
rickettsial diseases, 3rd ed. American Public Health
Association, New York.

Tevethia, S. S., and C. H. Cunningham. 1968. Antigenic
characterization of infectious bronchitis virus. J.
Immunol. 100: 793-798.

41.

42.

43.

44.

45.

39

Traver, M. I., R. L. Northrop, and D. L. Walker. 1960.
Site of intracellular antigen production by myxoviruses.
Soc. Exptl. Biol. Med. 104: 268-273.

Waterson, A. P., and J. G. Cruickshank, 1963. The effect
of ether on Newcastle disease virus. Z. Naturforsch. 18:
114-118.

Wheelock, E. F., and I. Tamm. 1961. Enumeration of cell-
infecting particles of Newcastle disease virus by the
fluorescent antibody technique. J. Exptl. Med. 113: 301-
316.

Wilner, B. I. 1969. A classification of the major groups of
human and other animal viruses. Burgess Publishing Co. ,
Minneapolis.

Zhdanov, V. M., N. B. Azadova, and L. V. Uryvayev. 1966.
Topography and dynamics of synthesis of structural pro-
teins of Newcastle disease virus. J. Bacteriol. 91:
1902-1906.

 

“11711111711171? 111111;”! 11111111111111 ti”