Characteristics of Parma Wallaby Herpesvirus Grown in Marsupial

423
J. gen. Virol. (1979), 45, 423-430
Printed h~ Great Britain
Characteristics of Parma Wallaby Herpesvirus Grown in
Marsupial Cells
ByJ. M 1 L L A R W H A L L E Y A N D C A R O L E. W E B B E R
School of Biological Sciences, Macquarie University, Sydney, N.S.W., Australia
(Accepted 30 May 1979)
SUMMARY
Parma wallaby herpesvirus (PWHV) has been characterized by a number of
properties. Electron microscopic examination of thin sections of infected cell
nuclei revealed virus nucleocapsids with various morphologies characteristic of
herpesviruses; enveloped particles were seen in cytoplasmic vacuoles and outside
cells. Negatively stained virus preparations from the medium of infected cell
cultures contained particles with the typical appearance of naked and enveloped
herpesviruses. The DNA of PWHV had a mean buoyant density in preparative
caesium chloride gradients of 1"712 g/ml, giving an estimated base composition of
51 ~o guanosine plus cytosine. The virus was able to replicate in all marsupial
cells, but not in most eutherian cells tested, and a single cycle of infection lasted
about 25 h. Infectivity was destroyed by a number of agents including lipid
solvents, acid pH and heat. The observed properties support the classification of
this virus as a new member of the herpesvirus family.
INTRODUCTION
The isolation of a probable herpesvirus from diseased Parma wallabies (Macropus parma)
by Finnie et al. (1976) is the first report of a herpesvirus affecting Australian marsupials. This
virus, provisionally referred to as Parma wallaby herpesvirus (PWHV) has been shown to
be experimentally transmissible to Parma wallabies, in which it caused a fatal disease
similar to that associated with the original isolation (I. R. Littlejohns, E. P. Finnie, H. M.
Acland & G. Maynes, personal communication). Evidence that PWHV or a closely related
herpesvirus is widespread among Australian marsupials came from a study of virus neutralizing antibody in sera from 242 animals in the wild and 116 in captivity (Webber & Whalley,
I978). Higher levels and frequency of antibody to PWHV were observed among captive
animals compared with those in the wild.
The aim of the present study was to characterize PWHV with respect to growth and host
range in celt culture, appearance in the electron microscope and buoyant density of virus
DNA.
METHODS
Cells and media. Primary cell lines of epithelial morphology were initiated from ear
biopsies from M. parma and maintained for approximately 15 passages. A primary cell
line of the marsupial mouse Antechinus rosamondae was initiated from body wall tissue by
Drs D. W. Cooper and P. Woolley, and maintained for over 30 passages. BHK2I cells were
supplied by Commonwealth Serum Laboratories (CSL), Parkville, Victoria, Australia.
Canine and feline kidney cells were kindly supplied by Dr M. Sabine, Department of
Veterinary Pathology, University of Sydney, Australia. SCB 18I (Wallabia bicolor) cells
oo22-I3t7/79/oooo-365o$02.00
~ 1969 SGM
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424
J. M. WHALLEY AND C. E. WEBBER
were a gift from Dr R. Moore, Cancer Institute, Melbourne, Australia. Other marsupial
cell cultures were grown from stocks held by Professor G. B. Sharman and Dr D. W.
Cooper. All cells were routinely grown in monolayer cultures in H a m ' s Fro medium (CSL)
containing IO to 20 ~o (v/v) foetal calf serum (FCS; Australian Laboratory Services or Flow
Laboratories) at 35 °C in an atmosphere of 5 ~o COs.
Virus stocks. The original stock of PWHV was kindly supplied by Dr I. R. Littlejohns
and E. M. Batty, Glenfield Veterinary Research Station, N.S.W., Australia. Herpes simplex
type I (HSV-I), Bryan strain, was a gift from Dr M. Cloonan, Prince of Wales Hospital,
University of New South Wales, Sydney, Australia. Bacteriophage T7 was a gift from Dr
M. Sabine. Stocks of PWHV were grown in M. parma cells, and HSV-I in B H K 2 I cells.
all virus stocks were twice plaque purified before use in this study.
Plaque assay. PWHV was assayed in 6o mm plastic dishes on confluent monolayer cultures of either M. parma, A. rosamondae or BHK2~ cells. The cells were pre-treated for at
least I h with 4#g/ml Polybrene (Aldrich Chemical Co., Milwaukee, Wis., U.S.A.), and virus
allowed to adsorb for 2 h, before overlaying with medium composed of H a m ' s FIo,
5 to I o % (v/v) FCS and 1"5% (w/v) methylcellulose. Plaques of rounded cells were read
microscopically 2 to 4 days p.i. The efficiency of infection in M. parma cells was approximately twice that in A. rosamondae cells and loo times that in BHK2I cells.
Treatment with physical and chemical agents. Virus samples from culture medium were
assayed before and after treatment with formalin, chloroform, ether, acid pH and temperatures of 37 and 56 °C (Rouzzo & Burke, t973), and were also exposed to cycles of
freezing and thawing or sonication.
Virus growth curve. PWHV stocks were prepared from infected M. parma cells by ultrasonication of cells and medium, followed by filtration through a o.45/~m pore size Millipore filter. Titres of virus prepared in this way were generally between I × io 7 and 2 × io v
p.f.u./ml. Maeropus parma cells and A. rosamondae cells were infected in suspension with
PWHV (m.o.i. 36: I) for I h in culture medium containing 2/~g/ml Polybrene. Infected cells
were washed four times with 6 ml of calcium and magnesium-free phosphate-buffered saline
(PBS-) and then plated on to 35 mm diam. plastic dishes in fresh culture medium and
incubated at 35 °C. At various times p.i. plates were chilled to o °C, cells scraped off the
plates and cells and media separated by centrifugation at zooo rev/min for l o min. Cells were
washed twice with fresh culture medium and the supernatants pooled and stored at 4 °C
until infectivity was measured by plaque assay. The cells were resuspended in fresh medium
and immediately before plaque assay were sonicated in a Branson sonifier at 5o W for
30 s.
Electron microscopy. Infected cells were prepared for electron microscopy by scraping
cultures off plastic dishes (when 7o~,, of the cells showed c.p.e.), centrifuging at 2ooo
rev/min for 5 min, fixing in 2~o glutaraldehyde and post-fixing in t ~o osmium tetroxide
prior to thin sectioning. Virus for negative staining was concentrated from infected cultures
by centrifuging clarified media at 27oooo g for 1 h and resuspending in a small volume of
buffer (o. l M-NaCI, o'o5 M-tris-HCl, o'oo5 M-EDTA, pH 8.o); droplets of virus suspension
were picked up on Formvar-coated grids and stained with 2~o ammonium molybdate.
Preparations were examined either in a Hitachi HS8 or a Philips 300 electron microscope.
Preparation ofnucleocapsids. Nucleocapsids from infected cells were prepared by modification of the method described by Kieff et al. 1971. Infected cells were scraped off Ioo mm
plastic dishes and centrifuged at 800 g for lo min at 4 °C. The cell pellet was washed with
cold PBS-, resuspended in RBS (o.ol M-NaCI, o.ool M-MgCI> o.o~ ~-tris-HC1, pH 7"5)
containing o ' 5 ~ (v/v) NP4o and left at 4 °C for lo min. The cell suspension was sonicated
for 3° s at 50 W and centrifuged at 8 o o g for Io min at 4 °C. The supernatant was layered
on IO to 5 o ~ sucrose gradients (in o'I5 M-NaCI, o.o2 M-tris-HCl, pH 7'5) and centrifuged
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Parma wallaby herpesvirus
425
for 3o min at 8t 5oo g in a Beckman SW27. I rotor. Fractions containing nucleocapsids
were pooled, pelleted by centrifugation at 27oooog for I h in the SW41 Ti rotor, and allowed
to resuspend in I M-NaCI, o.ol M-EDTA, o'o5 m-tris-HC1, pH 7"5, for I6 h at 4 °C- Such
preparations consisted mainly ( > 8o~o) of naked particles although some enveloped or
partly enveloped particles could also be seen on electron microscopic examination.
DNA extractions. P W H V D N A was extracted from nucleocapsids by incubation at
37 °C with 5o#g/ml RNase for 15 min, 30 min with autodigested pronase (5o#g/ml) in
2 % Sarkosyl (Ciba-Geigy Australia Ltd., Sydney) and I % sodium dodecyl sulphate,
followed by phenol extraction at 6o °C for 2 rain. The aqueous phase was further extracted
in chloroform/isoamyl alcohol (50: l) and dialysed against o.t M-NaC1, o.o~ M-EDTA,
o.oI M-tris-HCl, pH 7'4. Marker D N A either as D N A (Micrococcus luteus) or as whole
phage (T7) was added to nucleocapsid preparations and co-extracted with PWHV DNA.
Radioactive labelling. Virus D N A was labelled by growing virus in media containing
2o#Ci/ml *H-thymidine (The Radiochemical Centre, Amersham). T7 phage D N A was
labelled by growing infected E. coli B cells in broth containing 5/*Ci/ml ~2P-phosphorus
(Australian Atomic Energy Commission, Lucas Heights, Sydney, Australia). The phage
was purified by sucrose gradient centrifugation of a polyethylene glycol concentrate (Hayward & Smith, I972).
Analysis of caesium chloride gradients. D N A preparations were mixed with CsCI in TE
buffer (o-o 5 M-tris-HC1, o'oo5 M-EDTA, pH 8.o) to a density of 1"73o g/ml in a vol. of
approx. 4 ml and centrifuged in a Beckman 5o Ti rotor for 50 h at 16oooo g at 25 °C.
Fractions were collected from the bottom of the gradient through a fine tube and measurements made of refractive index, absorbance and radioactivity. Mean buoyant density values
for the D N A were obtained by analysing the data from I2 CsCl gradients, as follows: a
regression line was fitted to density versus fraction number for each gradient and each
fraction converted to density. Radioactivity was expressed as percentage total radioactivity
incorporated into DNA. Each gradient was grouped into o.oo2 g/ml density units (for
plotting percentage radioactivity against density) and the mean and standard error of the
percentage radioactivity for each grouping calculated.
RESULTS
Host range and growth in cell culture
Cell cultures from a wide range of marsupial species and a number of eutherian species
were tested for susceptibility to PWHV as evinced by the production of c.p.e, in which
infected cells typically became rounded up and subsequently detached from the surface of
the culture vessel The virus could also cause plaque formation in permissive cell cultures
overlaid with methylcellulose. Little evidence was found for syncytia formation or other
forms of cellular aggregation. P W H V was able to grow in cells of all the marsupial species
tested (Table I) but only in one of the eutherian cell types, namely the hamster line BHK2 I,
in contrast to the other hamster cell line BOR. In the course of these experiments, some
variation was observed in the time taken for c.p.e. (approximately 5 o ~ of cells affected)
to develop in different cells, for example from about 20 h for wallaby cells up to 12o h for
possum cells. The one-step growth cycle of P W H V in M. parma and A. rosamondae cells
was characterized by an eclipse period of between 7 and lo h, followed by a rapid increase
in the titre of infectious virus to a maximum level at about 25 h p.i. Production of virus in
the extracellular fraction generally paralleled the rise of virus in the intracellular fraction,
although a further increase was observed after 25 h which may have been due to release
of virus from dead cells. The overall yield of virus was 262 p.f.u, for each M. parma cell
and o. 4 p.f.u, for each A. rosamondae cell.
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J . M . W H A L L E Y A N D C. E. W E B B E R
T a b l e I. H o s t range in tissue culture cells
Permissive cells
Macropus parma (Parma wallaby)
M. eugenii (tammar wallaby)
M. rufus (red kangaroo)
M. giganteus (grey kangaroo)
M. rufogriseus (redneck wallaby)
M. rufogriseus x M. agilis
(redneck wallaby x agile wallaby)
M. r. robustus x M. rufus
(wallaroo × red kangaroo)
Macropus robustus erubescens x M. r. robustus (euro × wallaroo)
Antechinus rosamondae (marsupial mouse)
A. macutatus (marsupial mouse)
Sminthopsis crassicaudatus (marsupial mouse)
Petrogale Iongmani (rock wallaby)
P. godmani (rock wallaby)
Thylogale billardierii (Tasmanian pademelon)
Potorous tridactylus (potoroo)
Peramelidae (bandicoot)
Trichosurus vulpecula (brush tailed possum)
Vombatus ursinus (wombat)
SCB 18I (swamp wallaby)
BHK21 (hamster)
Non-permissive cells
HeLa
BSC (African green monkey)
Canine
Feline
BOR (hamster)
XC (rat)
Pseudomys novaehollandiae
(New Holland mouse)
Ornithorhynchus anatinus
(platypus)
Rattus Jbscipes (native rat)
(d)
Fig. I. Electron micrographs of Parma wallaby ear ceils infected with PWHV showing: (a) arrays
of nucleocapsids (NC) in cell nucleus; (b) nucleocapsids with various morphologies including
particles with dense cores (D), ring-like cores (R) and electron translucent centres (E): (c) cytoplasmic vacuoles (arrow) containing enveloped particles, microtubule-like structures (MT) are
also evident; (d) enveloped particle close to the outer cell membrane.
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Parma wallaby herpesvirus
,':
(b)
~:i~i!
i'ii:~:
~:iC
427
Fig. 2, PWHV negatively stained with 2 ~ ammonium molybdate. (a) Non-enveloped capsid and
(b) particle with capsid surrounded by distended envelope.
I
(a)
!
I
l
I
I
I
15
15
'7
|0
I
[
I
I
I
I I
(b)
i T7
M. luleus ill/ ~
>
o
10
x
©
?
E
"
0
5
10
15
20
Fraction number
25
5
:
i
I
I
I
I
I
I
1.73 1.72 1-71 1.70 1.69 1.68
Density of Cs('l (g/nil)
Fig. 3- (a) Separation of particles (mainly nucleocapsids) on zo to 50 % sucrose gradients showing
infected (©
©) and non-infected (O
0 ) Parma ear cells. (b) Composite caesium chloride
gradient of DNA from nucleocapsid preparation. (Bars show standard errors of mean ~o radioactivity.) Marker DNA positions are indicated by arrows.
Virus morphology
Infected cells
M. parma cells infected with P W H V were examined in thin sections by electron microscopy. Cell nuclei contained large numbers o f n o n - e n v e l o p e d particles (nucleocapsids) with
a m e a n diam. o f ~ to nm, m a n y of which were hexagonal in outline (Fig. I a, b). Occasional
enveloped particles were also seen in cytoplasmic vacuoles (Fig. I C). The virion shown in
Fig. I (d) clearly possessed 'spikes' projecting from or t h r o u g h an outer m e m b r a n e surr o u n d i n g a matrix or ' t e g u m e n t ' ( R o i z m a n & Furlong, I974) which in turn encircles the
nucleocapsid. The mean diam. o f these ' m a t u r e ' particles, including the spike region, was
I66 nm, with the nucleocapsid structure measuring I IO nm across.
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428
J. M. WHALLEY AND C. E. WEBBER
Negatively stained virus
The appearance of negatively stained PWHV is shown in Fig. 2. Under the conditions
used most particles appeared to lack an envelope; these 'naked' particles appeared hexagonal in outline and some internal structure could be seen in the capsid, including hollow
capsomeres (Fig. 2a). An occasional enveloped particle (Fig. 2b) was also seen with a distended envelope surrounding the capsid; surface projections (or spikes) were also visible
on part of the envelope.
Buoyant density and base composition of P W H V DNA
DNA was extracted from nucleocapsid preparations (Fig. 3a) and centrifuged to equilibrium in CsC1 along with marker DNAs. The results (Fig. 3 b) showthat PWHV DNA had a
mean buoyant density of 1"712 g/ml, corresponding to a base composition of 5I ~ guanine
plus cytosine ( G + C ; lift et al. t96 Q. Under the conditions used, the DNA appeared to be
present as two components in approximately equal proportions with buoyant densities of
I'7IO g/ml and I'714 g/ml corresponding to base compositions of 49~o and 5 3 ~ G + C ,
respectively.
Sensitivity to various treatments
The infectivity of PWHV was readily inactivated by lipid solvents, acid pH, formalin,
temperature of 56 °C and by cycles of freezing and thawing. The half-life at 37 °C was less
than t h.
DISCUSSION
The wide host range exhibited by PWHV in marsupial cells, in contrast to its inability to
grow in a number of eutherian cells, suggests that PWHV has evolved along with a marsupial host or hosts. In a previous report, Finnie et al. 0976) found that PWHV grew in
Parma wallaby, potoroo and pretty-faced wallaby (M. parryi) cells, but failed to grow in
certain bovine, murine or hamster cells. The host range studies on PWHV distinguish it
from those herpesviruses which are known to grow in the eutherian cell types tested (Darlington & Granoff, I973). The growth of PWHV, however, is not necessarily restricted to
marsupial cells, as shown by the susceptibility of BHKzI cells to the virus. Although PWHV
was unable to replicate in certain human cells, it is of interest that human herpesvirus I
(HSV-t) could produce c.p.e, in each of three marsupial cell types tested, namely M. parma,
A. rosamondae and a euro×wallaroo hybrid, as well as the Australian native mouse
Pseudomys novaehollandiae (unpublished observation).
The type of c.p.e, in cells infected by PWHV was essentially a lyric effect similar to that of
many herpesvirus infections in cultured cells (e.g. Darlington & Granoff, I973). During the
original isolation of PWHV, Finnie et al. 0976) described typical eosinophilic intranuclear
inclusions and occasional syncytia with giant cell formation. The intracellular growth cycle
of 25 h for PWHV in M. parma or A. rosamondae cells compares with I7 h for HSV-I
(e.g. Hoggan & Roizman, I959) and zo h for some other herpesviruses (Darlington &
Granoff, I973). The yield of PWHV in each M. parma cell of 262 p.f.u, was somewhat
lower than reported yields (48o p.f.u.) of HSV-I in cells (Hoggan & Roizman, 1959).
The electron microscopic examination of PWHV-infected cells revealed particles with
various morphologies which were characteristic of herpesviruses (reviewed by Watson,
I973, and Roizman & Furlong, 1974) and were consistent with different stages of virus
maturation. O'Callaghan & Randall (I976) proposed that partially dense capsids are precursors of the fully dense-cored capsids, which then become enveloped. In the present study,
the occurrence in the cytoplasm of PWHV particles with a tegument (Roizman & Furlong,
~974) and an envelope, is in accord with the belief that herpesviruses generally acquire
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P a r m a wallaby herpesvirus
429
their outer layers at the cell nuclear membrane (Darlington & Moss, ~969) either by modification of the existing membrane or by virus-induced synthesis of a modified nuclear
membrane. The cytoplasmic PWHV particles appeared to be in vacuoles, which may be
part of a specific transport mechanism between the nucleus and the outside of the cell
(Morgan et al. 1959; Schwartz & Roizman, 1969).
The mean diameters of enveloped PWHV particles (I66 nm), nucleocapsids (1IO nm),
ring-like cores (65 nm) and dense cores (26 nm) are within the range found for other herpesviruses and compare with values for HSV-I of I2O to I5O nm for enveloped particles, lO5
nm for nucleocapsids (Watson, 1973), 45 to 55 nm for ring-like cores and 35 to 45 nm for
dense cores (Roizman & Furlong, 1974). The distinctive appearance of negatively stained
PWHV particles, especially those with a distended envelope, are highly characteristic of
herpesviruses (e.g. Witdy et al. t96o; Watson, 1973).
Although analysis of preparative CsCl gradients does not provide absolute values (such
as those obtainable by analytical ultracentrifugation), the buoyant density and base composition of PWHV DNA is in the middle range of values for herpesviruses (Goodheart &
Plummer, I975). Some other herpesviruses with base compositions in this range are
cercopithecid herpesvirus t and 2 (B virus and SA6) with 51 ~oo G ÷ C and herpesvirus saimiri
with 5 o ~ G + C (from the summary by Roizman & Furlong, 1974). Despite this similarity
in base composition of DNA, these viruses are differentiated from PWHV by their host
range. The presence of two peaks of DNA in CsCI could be explained by the DNA being
extracted in the form of several discrete fragments with different base compositions. The
two bands were consistently observed and were unlikely to be due to the presence of two
strains of virus as the PWHV stocks were plaque-purified; it is also unlikely that one of the
peaks was due to a cellular D N A component, such as a satellite, since the DNA was obtained from nucleocapsid preparations separated on sucrose gradients. When uninfected
cells were similarly treated, no material corresponding to the nucleocapsid band was found,
also arguing against possible contamination by mycoplasma contributing to the DNA
profile. The presence of two peaks of DNA in CsCI gradients has been reported for a
number of herpesviruses and has been discussed by Goodheart & Plummer (I975). The
DNA of several murine cytomegaloviruses banded at 1.7t 7 g/ml and 1.722 g/ml when
present as fragments of one quarter length or less (Plummer et al. 1969; Mosmann &
Hudson, I973).
The data presented here, together with the initial description of clinical symptoms and
effects on cultured cells by Finnie et al. (1976), is evidence that this virus from a Parma
wallaby is a new member of the herpesvirus family, Herpetoviridae (Fenner, 1976) and
supports the suggestion (Webber & Whalley, 1978) that PWHV has evolved with a marsupial host, rather than having been recently transferred from another (eutherian) mammalian species.
The authors are grateful to Professor G. B. Sharman, Dr D. W. Cooper and colleagues
for access to their stocks of marsupial cells; thanks are also due to Dr D. Cockayne and
Mr. R. Wright (Electron Microscope Unit, University of Sydney) and Ms J. Gregory for
assistance with electron microscopy; to Ms D. Clucas and Mr R. J. Oldfield for photography. This research was supported by grants from the Australian Research Grants
Committee and from Macquarie University Research Grants Committee.
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