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Iranian Journal of Virology 2015;9(1): 7-12
©2015, Iranian Society of Virology
Original Article
Replication Kinetic of Infectious Laryngotracheitis Virus in
Embryonic Chicken Neural Stem Cell
Jamshidi Navroud Z1 , Shahsavandi S1* , Firouzi M2
1. Razi Vaccine and Serum Research Institute, Karaj, Iran
2. Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
Abstract
Background and Aims: Infectious laryngotracheitis virus (ILTV) infection causes a highly
contagious respiratory disease in poultry which leads to significant economic losses in the
poultry industry. The genomic and antigenic characteristics between virulent and vaccine
strains of ILTV are very similar. The ability of embryonated chicken neural stem (CNS) cell
for differential diagnosis of ILTV strains was also evaluated.
Materials and Methods: CNS cell cultures were inoculated with an ILTV vaccine stain and
a virulent strain at different multiplicity of infections (MOI). The replication and growth
kinetics of the viruses were evaluated by estimation of suitable time of infection, production
of virus infectivity titers, and amplification of ICP4 gene in collected samples.
Results: Based on PCR and virus titration assays, the ILTV strains were able to enter CNS
cells and replicate efficiently at all MOIs used. The virus titers and tracing of ICP4 data were
found to be similar, but marked cytopathic effect (CPE) was only detected in ILTV vaccine
stain infection as compared with virulent strain.
Conclusion: The neural stem cell had a higher growth potential and infectivity for ILTV
strains, which will be of advantage in differential diagnosis.
Keywords: Infectious laryngotracheitis virus, embryonic chicken neural stem cell,
Replication kinetic, cytopathic effect
Introduction *
I
nfectious laryngotracheitis virus (ILTV)
belongs to the Gallid herpesvirus-1, a
member in the genus Iltovirus, of the
Alphaherpesvirinae subfamily within the
family Herpesviridae. The dsDNA genome of
ILTV is approximately 150 kb composed of a
unique long (UL), a unique short (US) region,
and inverted internal (IR) and terminal (TR)
*
Corresponding author: Shahsavandi S, PhD in
molecular genetics. Razi Vaccine and Serum Research
Institute, Karaj, Iran.
Tel: (+98) 263 4570038-46
Fax: (+98) 263 4552194
e-mail: [email protected]
repeats. The virus causes ILT, a highly
contagious respiratory disease in poultry. The
disease is characterized by severe production
losses due to increased morbidity, moderate
mortality, decreased egg production, and
weight losses. ILT has been described in most
countries and remains a threat to the intensive
chicken industry. The controlling programs
focused on biosecurity measures, preventing
the spread of disease to other birds, and
vaccination to build up immunity in
susceptible birds. ILTV mainly transmitted
through chickens with acute upper respiratory
tract disease and latently infected (carrier)
fowls (1-4). According to the most generally
accepted lytic infection model, the virus entry
following fusion of the virion envelop to the
host cell plasma membrane. The linear viral
Iranian Journal of Virology, Volume 9, Number 1, 2015
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Replication Kinetic of Infectious Laryngotracheitis Virus in Embryonic Chicken Neural Stem Cell
DNA transported to the host cell nucleus and
rapidly circularized. Then the immediate early,
early and late genes are subsequently
transcribed. Viral capsid assembly and progeny
DNA encapsidation takes place in the nucleus.
The produced extracellular virions are in
infected cells (5, 6). The latent or nonproductive infection is established by
expression of latency-associated transcript
(LAT) at high abundance and repression of
lytic gene expression when the virus infects
sensory neurons (7).
The ILTV strains appear to be antigenically
homogenous based on virus-neutralization,
inmunofluorescence tests, and cross-protection
studies; however, they vary in virulence for
chickens (8). Polymerase chain reaction and
restriction fragment length polymorphism
(PCR-RFLP) analysis revealed that nine ILTV
genotypes are circulated in world (9, 10). The
genomic- and antigenic characteristics between
virulent and vaccine ILTV strains are very
similar (9-13). Despite the extended progresses
in molecular virology, differentiation of ILTV
strains of varying virulence is still an important
practical problem (8). All of the viruses are
widely propagated in chicken embryo kidney,
liver and fibroblasts and produced syncitia as
the marked cytopathic effect (CPE) of
herpesviruses (14). Moreover, the short life
span, high cost, and laborious preparation of
the primary cells make it desirable to establish
a new cell line of avian origin to replace. In
this study, we have assessed sensitivity of
embryonic chicken neural stem cell (CNS) to
ILTV infection using both virulent and
modified vaccine viral isolates. The dynamics
of infection at different multiplicity of
infections (MOI) was also evaluated.
Methods
Virus and cell
The locally isolated virulent ILTV strain
(provided by Dr M.M. Ebrahimi) and the
attenuated ILTV vaccine strain were used in
this study. CNS cells were grown in 25 cm2
flasks with Dulbecco’s modified Eagle
medium
(DMEM)
(Sigma-Aldrich)
supplemented with 10% fetal calf serum and
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Iranian Journal of Virology, Volume 9, Number 1, 2015
1% antibiotics (penicillin, streptomycin) and
incubated at 37°C in an atmosphere of 5%
CO2. Growth kinetic of the cell was estimated
up to four days. In case, CNS cell was seeded
at 1 × 105 cell concentration and total cell
numbers were counted each day. Data
represent mean values ± SD of three replicates.
Virus titration
Virus production was titered by plaque assays
using chicken embryo fibroblast cell. The
monolayer of the cell in DMEM was
transferred to 96-well microtiter plates and
incubated for 1-2 h. The collected virus
supernatants were serially diluted from 104- to
109 with growth medium. About 50 μl of each
dilution was transferred to the wells,
subsequently, DMEM containing 1% lowmelting-point agarose was added and the plates
were incubated at 37°C in 5% CO2 for 5 days.
Cell suspensions without virus served as
controls. The plaques were visualized by
staining with a 1% crystal violet solution in
20% ethanol. Virus titer was determined as
PFU per/ml and served for calculation of MOI.
Cell infectivity trial
Replication kinetic of ILTV strains in CNS cell
cultures was estimated up to four days.
Monolayers of the cell at concentration of
1×106 cells/ml were inoculated with each of
the ILTV strains at MOIs 0.01, 1.0 and 2.0.
After absorption for 1 h at 37°C, the inoculum
was removed and DMEM was replaced. For
each strain three different sets of culture flask
and also uninfected mock flask were
considered. Cells were monitored up to 96 hr
post-infection (hpi) every 24 h and inspected
for CPEs using an inverted microscope (Nikon
Eclips TS100, Japan). An infected monolayer
was removed from the flask and transferred to
microtubes for further processing. The viral
suspension was centrifuged to pellet cell
debris. The clear supernatant was collected
carefully and stored for further use. Cell
viability was estimated by Trypan blue 0.4%
(Sigma-Aldrich) following viral infection.
DNA extraction and PCR assay
Total DNA was extracted from infected cells
(GeneAll,
Korea)
according
to
the
manufacturers' instructions. Amplification of
the viral ICP4 immediately early gene was
Jamshidi Navroud Z et al
monitored using specific primers F:5'TTTGAGGGAGTGGGTCGAAA-3' and R:5'CCCGTACGGTGACACAGATA-3' (1500 bp
in length) and Taq DNA polymerase Master
Mix Red (Ampliqon, Denmark) in each virus
supernatants. The fragment was subjected to 30
cycles of 94°C for 30s, 57°C for 45s and 72°C
for 60s, and one cycle at 72°C for 10 min
following an initiation step at 94°C for 5 min.
Data analysis
The data were expressed as means±standard
deviation (SD). One-way ANOVA analysis
followed by Student's t test was used to
determine significant (P<0.05) differences in
the SPSS ver. 11 statistical software package.
Results
The CNS cell exhibits enhanced growth rates
production of virus and its infectivity titer was
48 h. The cell was exposed to the ILTV strains
over a range of input concentrations to
determine the relative susceptibilities to
infection. Cultures were compared for presence
of CPE, virus titers and detection of viral
ICP4. The cell was found to be susceptible to
attenuated ILTV strain and exhibited similar
CPEs in response to viral infection at different
MOIs. At 48 hpi cell rounding; at 72 hpi
syncytium formation; and finally cell
detachment from surface at 96 hpi were
observed, while no CPE was found when CNS
cells infected with the virulent strain (Figure1).
Suitable time for propagation of the viruses in
the cell was determined. Exponential growth of
CNS cells started after a lag phase of about
24 h, and cell concentrations were maximal at
1.42 × 106 cells/ml at 72 hpi. After the
Fig. 1. Cytopathogenicity of embryonated chicken neural stem cell to infectious laryngotracheitis virus vaccine strain
infection at MOI=1.0. A) Mock; un-infected cell, B) 48 hours post infection, and C) 96 hours post infection (200x
magnification).
Fig. 2. Metabolic activity of embryonated chicken neural stem cell following infectious laryngotracheitis virus
infection. Measurement of cell viability in the cells at different times post infection with a MOI=1.0. Data points are
mean ± SD of three independent experiments
and the accurate time of infection for
respective
maximum,
cell
concentrations
Iranian Journal of Virology, Volume 9, Number 1, 2015
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Replication Kinetic of Infectious Laryngotracheitis Virus in Embryonic Chicken Neural Stem Cell
Discussion
Fig. 3. Amplification of infectious laryngotracheitis
virus virulent strain at different hours post infection in
embryonated chicken neural stem cell.
declined and cells entered the death phase.
Based on this, time for propagation was set
between 48-72 hpi when the cells were in the
exponential phase. During this period, the cells
could serve as host for virus replication.
Significant (P<0.05) decrease in cell viability
was detected at the end of the trial that
confirmed the CPE data (Figure 2).
The infectivity titer of ILTV strains in CNS
cell was determined every 24 hpi. Increase in
viral titer was shown in both ILTV strainsinfected cells at all MOIs used. In both strains
the titers started to increase at 24 hpi and
continue more rapidly specially for vaccine
strain. In the later hours of culture, the viral
titer gradually decreased. Infectious virus was
produced in the supernatant at average
concentration of 4.2×107 ± 1.6×107PFU/ml by
vaccine strain and 3.6×107 ± 1.2×107 PFU/ml
by virulent strain at the highest MOI (each
experiment was repeated three times). An
observation of cells showed that the number of
released viruses was similar for the two ILTV
strains in which, lysis of the cells showed no
distinct difference between the strains. Virus
replication was also confirmed by detection of
ICP4 DNA in collected supernatants. CNS cell
cultures were infected with the vaccine and
virulent strains which were analyzed 96 hpi
for amplification of the target DNA sequences
(Figure 3). The PCR assay results were
correlated with the infectivity titer data.
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Iranian Journal of Virology, Volume 9, Number 1, 2015
Several primary cell cultures derived from
chicken embryo fibroblast, liver and
kidneywere susceptibleto ILTV strains with
different rates of virulence. The times for virus
isolation vary depending on the condition and
sensitivity of the cells and the amount of
infectious viruses.Generally, the permissive
cells did not support virus propagation with a
distinct CPE even at high infectivity rate (14).
Thus sensitive cell culture system is still
essential for the rapid and accurate differential
diagnosis ofILTVs. Here the ability on an
established chicken-origin cell for replication
of ILTV strains was studied.
In our experiments, both virulent and vaccine
ILTV strains replicated well in CNS cell but
only the vaccine strain produced marked CPE
typically developed as enlarged and rounded
cells and characterized by multi-nucleated
giant cells. The strain produced easily visible
CPE within the first 24 h of incubation and
also formed syncytia at the end of trial period.
This phenomenon is the result of expression of
viral fusion protein at the host cell membrane
during viral replication (5, 15). At different
MOIs, the vaccine strain was grown fairly well
in showing clear CPEs and also increasing in
titer. MOI is a parameter for viral infectivity in
a population of target cells. In another strain no
change in cell morphologyfollowing virus
infection was detected. Molecular assay is an
alternative approach for detecting viruses that
not produce CPE in cell culture. To address
whether the virulent strain-infected cells
yielded progeny viruses in lack of visible CPE
and syncytia, the presence of ICP4 DNA in
collected supernatants was assessed. The
immediate early gene plays critical role in
regulation of virus gene expression. The
synthesis of ICP4 as a major transactivator of
both early and late gene expression is essential
for viral replication. ICP4 is a 175 kD DNAbinding phosphoprotein which is abundantly
expressed in the infected cell (16, 17). The Nterminus of the protein was necessary for
stabilizing
the
transcription
initiation
machineryand activation of transcription
Jamshidi Navroud Z et al
cascade when interacts with the general
transcription factors TATA-binding protein
and TFIIB (18, 19). With each ILTV strain,
based on increase in viral titer and
amplification of ICP4, virus replicated
efficiently.
We demonstrated that ILTV strains with
various disease severities exhibited different
cellular infection characteristics and varied in
their capacities to infect the CNS cells
depending on the viral strain.Even though
different MOIs were used, a wide variation in
the abilities of the two ILTV strains to infect
CNS cell was found. It seems that interplay
between virus and cell has an important role in
CPE formation. Host factors responses have
been account for the virulent and vaccine ILTV
strains in chicken lung cells. Microarray
analysis has revealed that four host genes
include bone morphogenetic protein 2,
chromosome 8 open reading frame 79,
coagulation factor X, and neuropeptide Y were
expressed distinctly in ILTV strains infection,
however, 273 cellular factors modulate ILTV
infection (16, 20). A similar response was
determined by Ghadiri et al. (21) in the context
of host cellular factors and ILTV infection.
The present study has been focused with the
aim of subjecting an efficient cell line for
propagation and differential diagnosis of
ILTVs. The data presented here demonstrate
that CNS cells possess an enhanced potential to
produce ILTVs with increase in virus titer, as
well as exhibits marked CPE when infected
with an attenuated strain. Future studies with
an extended panel of more virulent ILTV are
under way to assess whether different rates of
virulence correlates with appearance of CPE in
the cell.
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