Download Replication of Avian Infectious Bronchitis Virus in African Green

J. gen. Virol.
0972), x6, 423-427
423
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Replication of Avian Infectious Bronchitis Virus in
African Green Monkey Kidney Cell Line VERO
( A c c e p t e a 12 June
I972)
Avian infectious bronchitis virus (IBV), a coronavirus, requires initial isolation in, and
adaptation to, chicken embryos (CE) before transfer to primary avian cell and chicken
tracheal organ cultures. These are the only presently known cell cultures in which IBV
replicates and produces cytopathic effects (c.p.e.) in serial passage (Estola, 1966; Cunningham, 197o). Monkey kidney cells have been reported (Fahey & Crawley, 1956; Steele &
Luginbuhl, 1964) to support relication oflBV without c.p.e, when first inoculated with virus
propagated in CE. Attempts apparently were not made to passage the virus in these cells.
Direct haemagglutination (HA) is not a normal property of IBV (Biswal, Nazerian &
Cunningham, 1966) or of the human coronaviruses (Kapikian, I969). However, human
coronaviruses OC 38 and OC 43 adapted to suckling mouse brain (McIntosh et al. 1969)
cause direct HA (Kaye & Dowdle, 1969) and produce syncytia and plaques in African green
monkey kidney and BSC-I cells (Bruckova, cited by Bradburne & Tyrrell, 1971).
The studies reported here were prompted by the possibility that IBV adapted to suckling
mouse brain (Simpson & Group6, 1959; Estola, I966, 1967; McIntosh et al. 1969; Kaye &
Dowdle, 1969; Bradburne, 197o) might also cause direct HA and replicate in African green
monkey kidney cell line VERO (Yasumura & Kawakita, 1963). This cell line was selected
because it was not one of those previously tested by the authors without success by direct
inoculation for replication of IBV, and similar lesions are produced by IBV in chicken
embryo kidney cells (CEKC) (Cunningham & Spring, 1965; Lukert, 1965; Akers &
Cunningham, I968) and by mouse-brain-adapted OC 38 and OC 43 in African green
monkey kidney cell cultures (Bruckova, cited by Bradburne & Tyrrell, 1971).
Stock viruses were IBV-4I (Massachusetts), 5th CE passage; IBV-42 (BEAUDETTE),hundreds
of CE passages; IBV-46 (CONNECTICUT), 7th CE passage; and IBV-42C, I35th CEKC
passage of IBV-42.
Litters of IO to 14, 5- or 6-day-old, suckling Swiss albino mice were used for each of three
serial intracerebral passages of the respective viruses. Encephalitic signs (Estola, 1966, 1967)
were produced by IBV-42 and IBV-42 C and virus was recovered in CEKC from the brains
of the mice on the day when signs were present (Table I). Neither 1BV-4I nor IBV-46
produced signs through three' blind' passages in mice and virus was not recovered in CEKC.
The VERO cells were supplied by Dr D. L. Croghan, Veterinary Biologics Division,
United States Department of Agriculture, Ames, Iowa, 5OOLO,as passage I29. The growth
medium was of Eagle's basal medium with non-essential amino acids supplemented with
5 ~ foetal calf serum (FCS), L-glutamine (I ml./1, of 2OO raM) (Grand Island Biological
Company (GIBCO), 3175 Staley Road, P.O. Box 68, Grand Island, New York, I4O72)and
antibiotics (lOO mg./ml, of pegicillin and streptomycin, 60oo mg./ml, of tylosin tartrate),
buffered to pH 7"4 with 7"5 ~ filtered sodium bicarbonate. Maintenance medium was the
same except that the FCS was!reduced to 2 ~oo.
Confluent monolayers of cells (Fig. I) in closed tubes, Leighton tubes with coverslips, and
6o x 15 mm. Petri dishes were used. Incubation was at 37 °. Petri-dish cultures were in an
atmosphere of 85 ~ relative humidity and 8 ~ CO2. The growth medium was decanted and
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424
Table I. Intracerebral serial passage* of lBV in 5- to 6-day-oM suckling mice. Virus
recovered on the day of eneephalitic signJr assayed in CEKC as p.f.u.[g, brain
Mouse passage
c-
I n o c u l u m for 1st p a s s a g e
Day
I B V - 4 2 6 × xo 5 E I D s 0 / m o u s e
I
2
3
IBV-4zC5 I x io 5 p.f.u./mouse
I
2
3
I
2
-I'8X
-I0 4
3'5 × 105
--
6 ' 6 X I0 6
5'4 X l o 6
1"9× I0 6
2. 7 x 105
--
5"0× IO 6
3"5 X I O 6
3
9.2
X IO 4
2 " 7 x I0 5
-8"OX
IO 4
I ' 4 X IO 6
--
* I n o c u l u m o-o2 m l . / m o u s c .
~' B r a i n s p o o l e d for r e s p e c t i v e v i r u s e s a n d s t o r e d a t - 9 0 °. A t t h e t i m e o f use as i n o c u l u m for t h e n e x t
p a s s a g e i n m i c e or for cell c u l t u r e s , t h e b r a i n p o o l w a s m a d e i n t o a l o % s u s p e n s i o n (w]v) w i t h P B S w i t h o u t
C a 2+, o r M g 2+, p H 7, + z % F C S + a n t i b i o t i c s .
the ceils were washed thoroughly with phosphate-buffered saline (PBS) without Ca z+ or
Mg z+, pH 7, + 2 ~ F C S + antibiotics. Tube cultures received o.z ml. inoculum. After incubation for 6o min., z ml. maintenance medium was added. Petri-dish cultures received o'5 ml.
inoculum. After 9o min. the inoculum was poured off and 4 ml. maintenance medium was
added if these cultures were to be used for propagation of virus. All cultures were examined
daily for c.p.e. Coverslip cultures were stained with May-Grunwald-Giemsa solution.
Maintenance medium was replaced with fresh medium on the 2nd or 3rd day.
Petri-dish cultures for plaque assay were overlaid with 4 ml. agar medium (equal parts of
z ~ agar gel (GIBCO) and growth m e d i u m + 3 ~o pancreatin) in place of maintenance
medium after the inoculum was poured off. A 2nd agar overlay was added at the 5th day.
Neutral red, o'5 ml. of a o.I ~ solution, was added 5 days later. The cultures were then
incubated for 45 to 6o rain. at 37 ° and 6o rain. at 4 ° before the plaques were counted.
V E R O cells were first inoculated with IBV-42 and 1BV-42C in the 3rd mouse brain
passages. Syncytia (Fig. 2) were present throughout the monolayer on the 6th day and all
cells were necrotic by the 7th day. Medium from the cells was the inoculum for the next
passage. On the 2nd and subsequent passages of the virus, syncytia detectable at 24 hr in
stained cultures and at 48 hr in unstained cultures increased in size and number and all cells
were necrotic by the 5th day. Medium from cell cultures 5 days after infection was the
inoculum used for successive passages of the virus and for plaque assays. Virus was in the
cytoplasm, but not the nuclei, of the syncytia and other infected areas of the monolayers
(Fig. 3) as observed by the indirect immunofluorescence method (Rodriguez & Deinhardt,
I96o) using anti-IBV-4I chicken serum and fluorescein conjugated anti-chicken horse
y-globulin (Roboz Surgical Instrument Co., Inc., 8IO I8th St., N.W., Washington, D.C.
2ooo6).
Plaques (Fig. 4) were slightly opaque and a to 3 ram. in diameter. Assays of IBV-42 from
the 4th, 7th, I3th and I4thpassagesin cells were 1.2 ×, 1.8 ×, 3"o ×, and 4"6 × Io4p.f.u./ml., respectively. Assay of IBV-42C from the 5th passage was 4.2 x ioap.f.u./ml, and 5"o × Io4p.f.u.[
ml. from the I3th passage.
Anti-iBV-41 chicken serum neutralized mouse-brain-passaged IBV-4z and IBV-42C by
plaque reduction in CEKC and VERO cell-passaged virus by plaque and c.p.e, reduction
in VERO cells.
There was no direct haemagglutination by either the mouse-brain- or cell-passaged
viruses.
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Fig. i. Uninoculated VERO cells. May-Grunwald-Giemsa stain.
Fig. 2. Syncytia produced by IBV-42 in VERO cells 2 days after infection. May-GrunwaldGiemsa stain.
Fig. 3. Immunofluorescence produced by IBV-42C in VERO cells.
Fig. 4. Plaques produced by IBV-42 in VERO cells Io days after infection. Neutral red stain.
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Fig. 5. Morphogencsis of IBV-42 C in VERO cells. Virus particles are intracytoplasmic and
appear to bud (a, b, e) into the cytoplasmic vacuoles.
Intracytoplasmic and extracellular virus (Fig. 5) had the typical morphologic features of
IBV (Becker et aL 1967; Nazerian & Cunningham, 1968; Uppal & Chu, 197o). Budding was
from the walls of the cytoplasmic vacuoles but not at the plasma membrane. Virus was
released extracellularly as the result of rupture of the vacuoles.
So far as the authors are aware, this is the first report of serial passage of IBV in a
mammalian continuous cell line.
This is Journal Article No. 5888 of the Michigan Agricultural Experiment Station.
Department of Microbiology and Public Health
Michigan State University
East Lansing, Michigan 48823, U.S.A.
U.S. Department of Agriculture
Agricultural Research Service
Regional Poultry Research Laboratory
East Lansing, Michigan 48823, U.S.A.
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C. H. CUNNINGHAM
MARTHA P. SPR1NG
K . NAZERIAN
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427
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