Replication of Infectious Laryngotracheitis Virus in a Quail Cell Line, QT-35

Author(s): William M. Schnitzlein and Deoki N. Tripathy

Source: Avian Diseases, Vol. 39, No. 3 (Jul. - Sep., 1995), pp. 528-531
Published by: American Association of Avian Pathologists
Stable URL: http://www.jstor.org/stable/1591805
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AVIANDISEASES39:528-531, 1995

Replication of Infectious Laryngotracheitis Virus

in a Quail Cell Line, QT-35
William M. Schnitzlein and Deoki N. Tripathy

Departmentof VeterinaryPathobiology,College of VeterinaryMedicine,

Universityof Illinois, Urbana,Illinois 61801
Received 3 October 1994
SUMMARY.The relativeresistanceof the quail fibroblasticcell line QT-35to infection by
infectious laryngotracheitisvirus (ILTV)was circumvented by the continual passaging of
infected cells in the presence of uninfected monolayers. Such virus-containingcells were
used to extend the infection to an otherwise refractoryquail liver cell line IQ1A,as well as
to a normallypermissivechicken liver cell line LMH,with nearlyequal efficiency.In contrast,
extracellularvirus that had been derived from either QT-35or IQ1A cells could not readily
infect either quail cell line, although LMHcells were still susceptible. Therefore,the block
to ILTVreplication in quail cells is probablyat the adsorption/penetrationstage.

RESUMEN.Replicaci6n del virus de laringotraqueitisen la linea celular de codornices

QT-35.
La resistencia relativade la linea celular de fibroblastode codornices QT-35 frente a la
infeccion con el virusde laringotraqueitis,fue evitadamedianteel pasajecontinuo de celulas
infectadasen presencia de monocopas no infectadas. Lascelulas que contenian el virus se
usaron para infectar la linea celular refractariade higado de codorniz IQ1A, lo mismo que
parainfectarla linea celular permisivade higado de pollo LMHcon practicamentela misma
eficiencia. Por el contrario,el virus extracelularderivado de las celulas QT-35 o IQ1A no
pudo infectarninguna linea celular, aunque la linea LMHera a6n susceptible. Por lo tanto,
el bloqueo de la replicaci6n del virus de laringotraqueitisinfecciosa es probablementedu-
rante el estado de absorci6n o penetraci6n.

Infectious laryngotracheitis virus (ILTV) is an MATERIALS AND METHODS

alphaherpesvirus that causes an infection in the
upper respiratory tract of poultry. This conta-
gious disease is manifested in reduced egg pro-
duction and even mortality. The causative agent
is routinely propagated in either the chorioal-
lantoic membrane of developing chicken em-
bryos or primary chicken epithelial cell cultures
(3). Because of the inherent variability of these
hosts, as well as the temporal constraints im-
posed by their use, avian cell lines have been
surveyed recently with respect to their suscep-
tibility to infection by ILTV (6,7). Results dem-
onstrated sustained replication of the virus in
cells of chicken hepatoma origin. Moreover, in
agreement with a previous report (2), ILTVwas
found to be capable of infecting a quail fibro-
blastic cell line, QT-35 (6). However, cytopath-
ogenicity was not observed after the second pas-
sage of virus in these cells, and virus genomes
could not be detected after six passages. Since
a second, distinct cell line could be of value in
future ILTVstudies, propagation of the virus in
QT-35 cells was reexamined in the present study.
Cell culture and virus. The Japanesequail fibro-
blast cell line QT-35 (5), the Japanesequail liver cell
line IQiA (1), and the chicken liver cell line LMH
(4) were maintained and infected with ILTVas de-
scribed (6). Afterinfection with cell-free virus or the
addition of virus-containingcells, quail cell mono-
layers were overlaid with growth medium, whereas
the serumcontent of medium coveringLMHcells was
reduced to 1%.Avirulentvaccine strainof ILTV,L608,
which originated at Schering-PloughAnimal Health
Corp.(Elkhorn,Neb.), was used throughoutthis study.
Establishment and maintpnanee of ILTV-in-
fected QT-35 cells. Initially, QT-35 cells in a 60-
mm tissue-culturedish were infected with LMHcell-
adapted ILTVat a multiplicityof infection of 1. After
5 days, when plaque formation was apparent, the
monolayerwas dissociated by treatmentwith trypsin,
and approximately15%of the cells were replated.A
similarpercentageof the resultantmonolayerwaspas-
saged 13 days later. Thereafter, the infection was
maintained by the application of approximately2%
to 15% of the dissociated cells to semi-confluent
monolayersof uninfected QT-35 cells at 4-to-10-day
intervals.

528

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 ILTVin QT-35 cells 529

DNA dot-blot hybrild7ation assay for ILTV rep-

lication. DNA was isolated at various times from
1 2 3 4
ILTV-infectedQT-35 cells during their sixth subcul-
turing and also from a corresponding uninfected
monolayer.An undiluted sample and three serialfive-
A
fold dilutions of each DNA extractwere assayed for
the presence of virus genomes by hybridizationwith
a radioactivelylabeled ILTVglycoproteinB (gB) gene
probe (6).
Plaque assays. Virusyields (plaque-formingunits
[PFU])were determined by infecting QT-35, IQ1A, B
or LMHcell monolayersin either 60-mm tissue-cul-
ture dishes or 6-well tissue-culture plates. Inocula
consisted of cell-free culturemedium,detached cells,
and adherent cells obtained at 3 days after the addi-
tion of dissociatedILTV-infected QT-35cells to a semi-
confluentQT-35cell monolayer.Detached cells were C
removed from the medium by centrifugationat 2000
rpm for 10 minutes and then resuspended in fresh
growth medium. Likewise,growth medium was add-
ed to adherent cells after their dissociation by tryp-
sinization. Inocula were applied to cell monolayers
immediatelyafterpreparation,except when adherent D
cells were firstdisrupted by freezing at -80 C with
subsequentthawing.In orderto visualizethe plaques,
monolayerswere stained with crystalviolet at 3 to 5
days postinfection (6).
E
RESULTS
Fig. 1. Dot-blot hybridizationassay of 32P-labeled
Propagation of ILTV in QT-35 cells. In the
ILTVgB gene with undiluted and three serial fivefold
original study (6), the frequency of productive dilutions (columns 1-4) of intracellularDNAsamples
ILTV infection in QT-35 cells was less than 0.01% from the following: QT-35 cells at 3 hr (row A), 24
of that in LMH cells. This low rate of infectivity hr (row B), 48 hr (row C), and 72 hr (row D) after
coupled with the limited replication of ILTV in addition of dissociated cells from the fifthpassage of
this host could account for the lack of sustained ILTV-infected QT-35cells, and uninfectedQT-35cells
replication of the virus in QT-35 cells (6). In (row E).
order to circumvent a direct interaction be-
tween virus and cell, an attempt was made to tected at the initiation of the infection and, pre-
passage the virus via intact, infected cells. After sumably, was primarily derived from the inoc-
the initial infection with ILTV, the cells were ulum consisting of dissociated cells. The amount
routinely maintained except for constant re- of viral DNA increased almost 25-fold by 2 days
plenishment with uninfected QT-35 cells. Dur- postinfection and then decreased approximate-
ing all subculturings, plaques formed by the ly twofold within the following 24 hours. This
second day after cell dissociation and exhibited reduction was probably due to cell lysis, since
giant cell formation and intracellular bridging the majority of the monolayer had either de-
on their perimeters. Although such cytopath- tached or exhibited a cytopathic effect by this
ology is characteristic of herpesvirus infection, time.
further verification of the continuous replica- Since QT-35 cells were strongly resistant to
tion of ILTV in QT-35 cells was necessary. infection by ILTVpropagated in LMH cells (6),
Therefore, during the sixth passage of infected the infectivity of the quail cell-adapted virus
cells, the presence of intracellular viral ge- was examined. Cells and medium, obtained at
nomes was temporally monitored by DNA dot- 3 days after the sixth subculturing of the ILTV-
blot hybridization (Fig. 1). ILTV DNA was de- infected QT-35 cells, were separately assayed

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530 W. M. Schnitzlein and D. N. Tripathy
Table 1. Infectivityof ILTVpropagatedin QT-35
cells.A
Cell line Source Yield (PFU)8
QT-35 Medium 1.0 x 10'
Detached cells 5.6 x 102
Adherentcells 7.3 x 103
LMH Medium 4.9 x 102
Detached cells 9.0 x 102
Adherentcells 4.6 x 103
AYieldswere determined 3 days after the 6th sub-
culturing of ILTV-infectedQT-35 cells onto an un-
infected monolayerof approximately2 x 106 cells.
BYields were normalized to represent the total
amountof infectious virus present in each fractionof
the subculture, as determined on the two cell lines.
for the presence of infectious virus by direct
application onto either QT-35 or LMH cell
monolayers (Table 1). The yields obtained for
virus present in adherent and detached cells
were similar in both cell lines. In contrast, virus
could barely be detected in the medium when
assaying in QT-35 cells, whereas the titer in
LMH cells was almost 50-fold higher.
Replication of QT-35 cell-associated ILTV
in avian cell lines. Previous attempts (6) failed
to infect a quail liver cell line, IQ1A, with ILTV.
Since those inocula had consisted of mature
virions as well as intracellular virus that had
been released by prior freezing, the procedure
was repeated using cell-associated ILTV. Ad-
herent QT-35 cells, representing the 13th pas-
sage of ILTV-infected cells, were dissociated and
applied to monolayers of IQ1A, LMH, and QT-
35 cells. Similar titers were obtained, regardless
of the indicator cell line used (Table 2). How-
ever, after disruption of the infected cells due
to placement at -80 C, the amount of infectious
virus apparently decreased approximately two
orders of magnitude based on assays in either
IQ1A or QT-35 cells. In contrast, no decrease
in virus viability was detected when assaying in
LMH cells.
DISCUSSION
Extracellular ILTVcannot readily infect quails
(8) or cell lines derived from quails (6,7). In
the case of QT-35 cells, this deficiency could
not even be overcome by propagating the virus
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Table 2. Tropismand stabilityof ILTVpropagated
in QT-35 cells.A
Cell line FrozenB Titer (PFU/ml)c
QT-35 - 4.3 x 103
+ 7.8 x 10'
IQ1A - 6.2 x 103
+ 5.7 x 101
LMH - 4.3 x 103
+ 6.8 x 103
ATiterswere determined 3 days after the 12th sub-
culturing of ILTV-infectedQT-35 cells onto an un-
infected monolayerof approximately4 x 106 cells.
BWhenrequired, ILTV-infectedQT-35 cells were
disrupted by placement at -80 C.
CTitersrepresent infectious virus present in adher-
ent cells, which had been resuspendedin 2 ml growth
medium following dissociation.
in the homologous host (Table 1). This inability
is not directly due to a block in virus replication,
since quail cell lines can be indirectly infected
by contact with ILTV-containing QT-35 cells. In
fact, an IQ1A cell line infected with ILTV was
initiated in this manner and then maintained
by continuous passaging in the presence of un-
infected IQ1A cell monolayers. Titration of vi-
rus present in adherent IQ1A cells (fifth passage
of infected cells) produced results similar to
those obtained using the ILTV-containing QT-
35 cells (Table 2) in that freezing resulted in
decreased virus infectivity only when determi-
nations were made using QT-35 or IQ1A cells
and not when using LMH cells (unpublished
data). These reductions in virus titer probably
reflect the physical disruption of the majority of
the infected cells. In contrast, the seeming lack
of effect on virus viability when titering in LMH
cells can be attributed to the ability of at least
some of the released virus to still infect this
host.
The requirement for cell contact to initiate
and/or maintain ILTV infection in quail cell
lines implies that few, if any, receptors for this
virus exist on the surfaces of these cells. Alter-
nately, a subsequent step in the virus replicative
cycle, such as penetration, may be blocked. It
is unclear how any extracellular virus is able to
infect QT-35 cells. The relatively low frequency
of this event (6), even when the virus has been
propagated in QT-35 cells (Table 1), could re-
flect limited cell-virus interactions. Such rare
 ILTV in QT-35 cells 531

events must occur between QT-35cells and ex-
tracellularLMHcell-adapted ILTV,since intra-
cellular infection can be transferredonly to ho-
mologous LMHcells and not to the heterolo-
gous quail cells (unpublished data).
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ACKNOWLEDGMENTS
This work was supported by USDA grant AG-87-
Schering-Plough Animal Health Corp.

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