Establishment of persistent infection with foot-and-mouth disease virus in BHK-21 cells
Establishment of persistent infection with foot- and-mouth disease virus in BHK-21 cells
Xuan Huang 0
Yong Li 0
Hui Fang 0
Congyi Zheng 0
0 State Key Laboratory of Virology, College of Life Sciences, Wuhan University , Wuhan 430072 , China
Background: Foot-and-mouth disease virus (FMDV) is able to cause persistent infection in ruminants besides acute infection and disease. Since the mechanisms of viral persistence and the determining factors are still unknown, in vitro systems help explore and reveal mechanisms of persistence in vivo by providing useful models for the study of RNA genome mutations and evolution. Ammonium chloride, a lysosomotropic agent that raises intralysosomal pH, reduces the yield of FMDV during infection of BHK-21 cells. Results: The persistent infection with FMDV serotype O in BHK-21 cells was selected and established readily after treatment of ammonium chloride that acts primarily on the cells. Intact virions were observed located inside the endosomes. Viral genome RNAs and specific proteins were detected in the persistent cells to validate the establishment of viral persistence. Infection of the persistent viruses could not form plaques in host cells but virulence was enhanced. Basing on analysis and comparison of cDNA sequences of resident viruses and wild type viruses, 15 amine acid mutations were found, all of which were located in nonstructural proteins rather than in structural proteins. Conclusions: Therefore, persistent infection of cell cultures with FMDV is successfully established with some distinctive features. It would be worthwhile to further investigate the mechanisms of viral persistence.
Virus replication is harmful or pathological for interfering
host cell functions , but some viruses can reside
persistently in their host cells without causing any obvious
pathological changes, and keep the way to coevolve [2-5].
Foot-and-mouth disease virus (FMDV), the aetiological
agent of foot-and-mouth disease (FMD), which belongs to
the Aphthovirus genus of the Picornaviridae family ,
usually causes an acute, systematic infection of
clovenhooved animals and often produces a persistent
noncytocidal infection of ruminants . So far, seven distinct
serotypes (A, O, C, Asia1, and South African Territories1,
2, and 3) have been identified with a wide range of
subtypes within each serotype . The FMDV genome
consists of a single plus-sense stranded RNA of
approximately 8.5 kb in length, which contains a single open
reading frame flanked by two non-coding regions and a small
viral protein VPg linked covalently to the 5’end [9,10].
Replication of FMDV genome RNA occurs via a
complementary negative strand RNA. Negative strands were
detected in hundredfold lower concentration than positive
strands in acutely infected cells, suggesting that each
negative strand may serve as a template for the synthesis of
many positive strands . Its capsid is composed of 60
copies of each of the four structural proteins VP1-VP4 to
form an icosahedral symmetry, in which some viral
determinants are involved in the establishment of persistent
Among all the picornaviruses, FMDV is the most
sensitive to acidic conditions which may in fact enable infection
in vivo but have a detrimental effect on the virus in vitro
[13,14]. It has been speculated that the 140S virion of
FMDV probably breaks down into 12S pentameric
subunits upon entering an acidic endosome, releasing the
RNA . The weak lysosomotropic bases that diffuse
into acidic endosomes increase the endosomal pH, which
result in inhibition of virus infection by human rhinovirus
and FMDV [15-17]. Previous studies showed that some
lysosomotropic agents that block acidification of
endosomes, inhibit FMDV infection, indicating that genome
RNA release is dependent on endosomal acidification
[13,18,19]. Ammonium chloride, one of the weak
lysosomotropic bases that inhibit the pH decrease in endosomes
and lysosomes, where the proteolytic processing of viral
outer capsid proteins by acid-dependent cellular proteases
may occur, can block an early step in infection by intact
virions [20-23]. Canning and Fields have demonstrated
experimentally that the ammonium chloride can reduce
the yield of reovirus and help establish persistent infection
rapidly in mouse L cells .
To explore and improve our understanding of the
mechanisms of foot-and-mouth disease virus persistent
infection, a useful model of in vitro persistence is needed.
De la Torre and his colleagues have established persistent
FMDV serotype C (FMDV C) infection by growth of
BHK-21(c-13) cells or IBRS-2(c-26) cells that survived
standard cytolytic infection with FMDV until massive cell
detachment [25,26]. To avoid possible interactions
among cells, we selected persistent infection from one
cell clone that carried viruses using single cell techniques.
Unfortunately, the expected persistence was not observed
through several attempts by growth of BHK-21 cells that
survived cytolytic infection with FMDV serotype O
(FMDV O), the severe serotype that is responsible for
outbreaks in large areas of the world. However, the
persistent infection with FMDV O in BHK-21 cells can be
rapidly selected and established via 20 mM ammonium
chloride treatment of infected cells. This in vitro model
showed that FMDV persistence was established by
enhancing the resistance to virus infection at the cellular
level facilitated by inhibition of endosome-lysosome
system acidification with the aid of ammonium chloride. In
addition, we reveal that the persistent viruses possess
some distinctive features such as fixed locations,
increased virulence, steady replication efficiency in late
passages, unimpaired internalization ability, and amino
acid replacements in nonstructural proteins.
Detection of viral genome RNAs in acutely infected
BHK21 cells maintained in the presence and absence of
To investigate the effect of ammonium chloride on the
virus yield post-infection, BHK-21 cells in 12-well plates
were infected with FMDV and then maintained in 5% FBS
MEM supplemented with different concentrations of
ammonium chloride. Viral genome RNAs were detected
and quantified every 6 h from 0 h post-infection (h.p.i.) till
30 h.p.i. to explore the influences of weak bases on virus
infection. The amount of viral RNAs increased sharply
and reached a relative peak (exceeding 109 copies/105cells)
at 30 h.p.i. without ammonium chloride. However, it was
shown in Figure 1 that the presence of ammonium
chloride reduced the accumulations of virus replication by 1-2
magnitudes at all time points detected. Moreover, the
extent of suppression on FMDV infection depended on
the concentrations of ammonium chloride. The results
Figure 1 Effect of ammonium chloride on the yield of FMDV
genome RNAs in BHK-21 cells. 5 × 105 BHK-21 cells were infected
with FMDV at a m.o.i. of 0.01 PFU/cell and maintained in 5% FBS
MEM without (0 mM) or with different concentrations of
ammonium chloride (10, 20, 30, 40 mM ), respectively. Viral genome
RNAs were extracted and performed on qRT-PCR every 6 h from
0 h.p.i. to 30 h.p.i. The unit of Y axis is Log10 RNA copies per
105 cells, which were evaluated and corrected using qRT-PCR for
quantitation of GAPDH. All the cell samples were tested in triplicate.
demonstrated that ammonium chloride can reduce the
yield of FMDV during infection of BHK-21 cells.
Determination of the optimal concentration of
The viral RNA positive rate (the number of single-cell
samples containing viral genome RNAs/the number of
single-cell samples tested) and the number of cells
survived in FMDV infection maintained in the presence and
absence of ammonium chloride were tested and calculated
to explore the feasibility of establishing viral persistence
with the help of weak bases and the optimal
concentration. Confluent BHK-21 cell monolayers (106 cells)
infected with FMDV at m.o.i. of 0.01 PFU/cell resulted in
cytopathic effect and massive cell detachment at 48 h.p.i.
However, some cells remained attached to the surface, and
when provided with fresh medium, they could survive and
grow. The number of cells survived (an average value of
three independent assays) during FMDV infection
maintained in the absence and presence of ammonium chloride
(10, 15, 20, 30, 40 mM) differed dramatically (Figure 2A,
p < 0.0001). The survival rate reached a peak in the
presence of 30 mM ammonium chloride. Meanwhile, the
survived cells were rinsed with PBS three times and then
trypsinized to yield the cell suspension. In total, 300
single-cell samples were isolated from six different cell
suspensions (50 samples per one cell suspension), lysed
and then run on one-step qRT-PCR assays to detect
Figure 2 Treatment of BHK-21 cells with different concentrations of ammonium chloride. 1 × 106 BHK-21 cells were infected with FMDV
at a m.o.i. of 0.01 PFU/cell and maintained in 5% FBS MEM without (0 mM) or with different concentrations of ammonium chloride (10, 15, 20,
30, 40 mM ) for 48 h, respectively. After removing the alkalescent mediums, infected cells kept in fresh MEM with 10% FBS for 24 h followed by
single-cell clone selection and cell counting. (A). The number of cells survived the cytolytic infection maintained in the absence or presence of
ammonium chloride. (B). The viral genome RNA positive rates of BHK-21 cells survived. 50 single-cell samples were isolated from each cell
suspension survived (300 in total), lysed and run on one-step qRT-PCR assays for detection of viral positive-stranded RNAs. The assay was
repeated once. (C). The number of positive cells under the treatment of different concentrations of ammonium chloride.
the viral genome RNAs (positive-stranded RNAs). The
assay was repeated once. The highest positive rate was
approximately 25% with the treatment of 20 mM
ammonium chloride (Figure 2B). In conclusion, the number of
viral RNA positive cells survived was shown in Figure 2C.
Approximately 150 viral RNA positive cells out of 106
cells survived in virus infection maintained in the presence
of 20 mM ammonium chloride, a number that is
significantly more than when ammonium chloride was absent
(only one positive cell out of 106 cells). Therefore, the
establishment of persistent infection with FMDV in
BHK21 cells was feasible and the concentration of 20 mM
ammonium chloride was optimal and chosen for selection
of viral persistence.
Selection of persistent infections with FMDV in BHK-21 cells
After 48 h maintenance in 20 mM ammonium chloride,
infected cells were allowed to grow in fresh medium for
24 h without the weak base pressure when single cells
were isolated and put onto 96-well plates (one cell/well).
After 7-12 days incubation at 37°C, single-cell clones
were passaged routinely and then viral RNAs were
detected using qRT-PCR. 17 positive cell clones were
obtained in three independent experiments, named as
BHK-Op cells (with a number appended to “Op”
indicating passage times, such as BHK-Op15). One of the
persistent single-cell clones was selected randomly for virus
identifications, while the rest of the positive cell clones
were frozen in liquid nitrogen.
Electron microscopy observations of FMDV particles
BHK-Op48 cells were selected randomly for electron
microscopy assays to confirm the establishment of
persistent infection. FMDV particles were observed successfully
in BHK-Op cells, which dispersed in the cytoplasm and
were located in some small endosomes with a diameter
about 25 nm (Figure 3A). There were less virions observed
in the persistent cells compared to the acutely infected
cells (Figure 3B) and locations of viral particles differed in
the two cells. As shown in Figure 3B, virions in acutely
infected cells were located mainly in endosomes close to
the nucleus in large quantities. However, virions in
persistent cells were located in some endosomes that dispersed
in the cytoplasm in very small quantities (not more than
10, usually 2 or 3). The results suggested that ammonium
chloride may play an important role in the establishment
of persistent infection in our assays for ammonium
chloride helped prevent the virions releasing after entering
endosomes and reduced the yield of viruses via raising the
pH of endocytic organelles.
Morphological features of BHK-Op cells
Compared to normal BHK-21 cells (Figure 4A), there is
not any visible change in the morphological appearances of
BHK-Op cells both in early stages (BHK-Op8, Figure 4B)
and late stages (BHK-Op48, Figure 4C). Although
BHKOp cells coexisted with viruses, they did not manifest
cytopathic effects which were observed in acutely infected cells
(Figure 4D), suggesting that the persistence of FMDV does
not change morphological features of BHK-21 cells and
has no influence on the cell growth and proliferation.
Detection of viral proteins
Western blot analysis detected FMDV specific proteins
3D (viral RNA dependent RNA polymerase) and 3CD (a
protein precursor consisting of two different proteins
3C and 3D) in BHK-Op48 cells and acutely infected
BHK-21 cells (30 h.p.i., positive control) (Figure 5). The
Figure 3 Electron microscopy (EM) observation of FMDV particles in BHK-Op cells. A total of 5 × 106 BHK-Op48 cells and BHK-21 cells
infected acutely with FMDV (48 h.p.i.) were collected, fixed and prepared for EM observations. (A). The 48th passage of BHK-Op cells (BHK-Op48).
The arrows point out the viral particles. (B). BHK-21 cells infected acutely with FMDV. The arrow marks the viral particles.
results showed that the amounts of 3D and 3CD
proteins in the persistent cells were less than that of the
proteins in positive cells.
Quantitation of FMDV positive-stranded and
Viral RNAs of BHK-Op cells (BHK-Op12, 18, 24, 30, 36,
42, 48, 54, 60, 66) were extracted to perform on the duplex
qRT-PCR assays . The results shown in Figure 6A
indicated that the amount of FMDV positive strands
ranged from 107 to 109 copies per 100000 cells (the number
of cells was evaluated and corrected using qRT-PCR for
quantitation of house-keeping gene GAPDH, data not
shown), with a relative peak at passage of 36. The ratios of
positive strands to negative strands increased gradually in
the early stages and reached a climax of 973 at
BHKOp42, and then traced back to low values in the late stages
(Figure 6B), suggesting that viruses and host cells might be
struggling for a balance in order to coexist before passage
36. It was speculated that the viruses and host cells were
struggling for balance in early passages and the persistent
viruses lived harmoniously with host cells with steady
Figure 4 Morphological observation of BHK-Op cells. (A).
BHK-21cells. (B). BHK-Op8 cells. (C). BHK-Op48 cells. (D). BHK-21 cells
acutely infected with FMDV.
levels of viral replication in late passages after a rapid
decrease of the viral loads.
Detection of cell viability in the presence of ammonium
To determine whether the cells were affected first by
ammonium chloride, MTT assays were carried out to
show resistance of cells to the weak bases. As shown in
Figure 7, when treated with 20 mM ammonium chloride
for 24 h and 48 h, the survival probabilities of BHK-21
cells were not more than 45%, indicating that the presence
of ammonium chloride had a serious influence on cell
growth. However, the survival probabilities of BHK-Op
cells exceeded 75% which were much higher than that of
BHK-21 cells (p < 0.001). In conclusion, the persistent
cells which were selected and obtained with the help of
ammonium chloride were more resistant to ammonium
Figure 6 The amounts of viral RNAs and the levels of viral RNA
replication dynamics in different passages of BHK-Op cells. Viral
RNAs of BHK-Op12, 18, 24, 30, 36, 42, 48, 54, 60, 66 were extracted
to perform on duplex qRT-PCRs. (A) Quantitation of viral positive
strands and negative strands. (B). The ratios of positive-stranded
RNAs to negative-stranded RNAs.
chloride compared with normal cells, implying that
ammonium chloride acted on the host cell. Furthermore,
the facts that the survival rates of late passages of
BHKOp cells (BHK-Op48) were much higher than that of early
passages of BHK-Op cells (BHK-Op8) indicated that the
effects of ammonium chloride on the host cell retained
and may be enhanced through cell passage.
The influences of ammonium chloride on the growth of
BHK-Op8 cells, BHK-Op48 cells and acutely infected
BHK-21 cells were frozen at -80°C for at least 1 h, and
then thawed at 37°C, respectively. Freezing and thawing
were repeated for a total of three cycles for lysis of cells
and release of viruses. After 30 min of centrifuge at 3000 g,
4°C, the supernatants were filter-sterilized with 0.22
μmfilter and collected as virus suspensions (The viruses in
BHK-Op8 cells, BHK-Op48 cells and acutely infected
Figure 5 Western blot analysis for detection of viral proteins
3D and 3CD. Western blot analysis detected FMDV specific proteins
3D (viral RNA dependent RNA polymerase) and 3CD (a protein
precursor consisting of two different proteins 3C and 3D) in
BHKOp48 cells and BHK-21 cells infected acutely (30 h.p.i., positive
control). b-actin was included as control of protein loading and the
number of cells.
Figure 7 Cell viability of BHK-21 cells and BHK-Op cells in the
presence or absence of 20 mM ammonium chloride. MTT assays
were used to determine the viability of BHK-21 cells and persistent
BHK-Op cells treated with 0 mM (negative control for this assay) or
20 mM ammonium chloride. Cell survival rate was measured at
three different time points: 24 h, 48 h maintenance in ammonium
chloride and 48 h maintenance in ammonium chloride then in fresh
10% FBS MEM for 24 h. The results are detected from two
BHK-21 cells were named as FMDV-Op8, FMDV-Op48
and FMDV wild type.). The virus suspensions were
performed on RNA extraction using Trizol LS reagent
according to the manufacturer’s instructions and then run on
qRT-PCR for quantitation of viral RNAs.
Confluent BHK-21 cells in 12-well plates were infected
with FMDV wt, FMDV-Op8 and FMDV-Op48 at equal
amounts of viral RNAs. After a 1 h absorption period, the
inoculum was removed and 1 ml fresh medium was added
(with or without 20 mM ammonium chloride). To address
the influences of ammonium chloride on the growth of
persistent viruses, cell samples were collected every 6 h
from 0 h post-infection (h.p.i.) till 30 h.p.i. and then total
RNAs were extracted and quantified.
As shown in Figure 8, the presence of 20 mM
ammonium chloride reduced the yield of FMDV wt at all time
points detected. Likewise, the viral RNAs in BHK-21
cells infected with FMDV-Ops incubated with 20 mM
ammonium chloride were 10-fold less than that of cells
maintained free of ammonium chloride. The inhibition
rates of virus yield in wild type viruses and persistent
viruses were similar. In conclusion, infection of wt
viruses and persistent viruses were both sensitive to
ammonium chloride treatment. In other words,
FMDVOps did not gain the resistance to ammonium chloride.
Infectivity and virulence of FMDV-Ops
Plaque assays and TCID50 assays were designed to
compare the infectivity and virulence of FMDV-Ops
with FMDV wt. As mentioned in the text, suspensions
of FMDV wt, FMDV-Op8 and FMDV-Op48 were
filter-sterilized and quantified using qRT-PCR. Both
plaque assays and TCID50 assays were carried out using
equal amount of viral genome RNAs of the three
viruses for virus infection. The TCID50 value of the
three viruses were calculated every 12 h from 36 h.p.i.
Neither FMDV-Op8 nor FMDV-Op48 infection can
form plaques in BHK-21 cells after 3 d maintenance in
the semi-solid mediums while infection of FMDV wild
type (FMDV wt) resulted in plenty of large plaques
under the same condition (Figure 9A). However, the
fact that TCID50 of FMDV-Op8 (10-5.375) and
FMDVOp48 (10-5) were smaller than that of FMDV-wt
(10-4.375) at 72 h.p.i. implied that the virulence of
FMDV-Ops was enhanced compared to that of wild
type viruses (Figure 9B) although the persistent viruses
lost the ability to form plaques.
Figure 8 Growth of FMDV wt and FMDV-Ops in BHK-21 cells
maintained in the presence and absence of 20 mM ammonium
chloride. Confluent BHK-21 cells in 12-well plates were infected
with FMDV wt, FMDV-Op8 and FMDV-Op48 at equal amounts of
viral RNAs and then maintained with or without 20 mM ammonium
chloride. Cell samples were collected every 6 h from 0 h
postinfection (h.p.i.) till 30 h.p.i. and then total RNAs were extracted and
quantified via qRT-PCR. All the cell samples were tested in triplicate.
Figure 9 Infectivity and virulence assays of FMDV-Ops and wt.
(A) Plaque assays. Plaque assays were designed to compare the
infectivity of FMDV-Ops with wild type. Confluent BHK-21 cells in
6-well plates were infected with FMDV wt, FMDV-Op8 and
FMDVOp48 at equal amounts of viral RNAs and were overlaid with 3 ml
maintenance medium (2% FBS) containing 0.8% purified agarose
and incubated for 3d. Plaques were stained with 1 ml 1% crystal
violet in 10% paraformaldehyde. A. FMDV wt. B. FMDV-Op8. C.
FMDV-Op48. (B) TCID50 assays. TCID50 assays were designed to
compare the virulence of FMDV-Ops with wild type. The TCID50
value of FMDV-Ops and wt were calculated every 12 h from 36 h.p.i.
till 72 h.p.i.
BHK-21 cells were infected with FMDV-Op8,
FMDVOp48 and FMDV wt at equal amount of viral genome
RNAs with different absorption periods (1 h, 2 h and 4 h).
Both the supernatants and cell samples were collected and
run on qRT-PCR for detection of viral RNAs. The results
in Figure 10A showed that the amounts of viral RNAs in
FMDV wt infected cells and FMDV-Op infected cells were
similar to each other after 1 h (2 h) absorption whereas
the amounts of viral RNAs in FMDV wt infected cells
were higher than 10-fold of that in FMDV-Op infected
cells at 4 h.p.i. (after 4 h absorption), indicating that all the
three viruses could bind to the cell surfaces and only the
wild type viruses began to replicate within 4 h.p.i.
Moreover, the FMDV wt rather than FMDV-Ops released
viruses to the supernatant at 4 h.p.i.(Figure 10B) which
was consistent with the results in Figure 10A.
Mutations in the genome of FMDV-Op
To evaluate the spectrum of mutations in the persistence
of FMDV, we analyzed and compared the sequences of
full cDNA clone of FMDV wt and FMDV-Op48. Protein
sequence comparison and analysis of FMDV-wt and
FMDV-Op48 revealed that all the amine acid mutations
were located in nonstructural proteins rather than
structural proteins (Table 1). No mutations were found in
structural proteins, which was consistent with the results
of virus internalization assays, indicating that the
recognition of cell receptors of the persistent viruses were not
influenced and the persistent viruses can bind to the cell
surfaces and enter into the host cells as wild type viruses
Foot-and-mouth disease is one of the most highly
contagious diseases, which has been deemed as the most
important constraint to international trade in animals
and animal products . Its causative agent, FMDV, is
able to cause persistent infection in ruminants in
Figure 10 The internalization assays of FMDV-Op8,
FMDVOp48 and FMDV wt. (A) Quantitation of cell samples after 1 h, 2 h
and 4 h absorption of three viruses. (B) Quantitation of the
supernatants from each cell sample. All the cell samples and
supernatants were tested in triplicate.
Glu® Ala (aa 1174)
Val® Leu (aa 1389)
Pro® Thr (aa 1394)
Asn® Ser (aa 1418)
Ile® Val (aa 1498)
Thr® Asn (aa 1522)
Lys® Val (aa 1535)
Asp® Asn (aa 1563)
Leu® Arg (aa 1584)
Lys® Asn (aa 1642)
Glu® Gln (aa 1698)
Asp® Asn (aa 1753)
Gly® Arg (aa 1779)
Glu® Lys (aa 2009)
Val® Leu (aa 2187)
Mutation types and positions
* The nucleotide sequence of wild type virus (serotype O, Akesu/58/2002) was
used as reference, GenBank accession no. AF511039.
addition to causing acute infection and disease, which
makes control efforts even more costly. Since the
mechanisms of viral persistence and the determining
factors are still unknown, in vitro systems (viral
persistence in cultured cells) may help explore and reveal
mechanisms of persistence in vivo by providing useful
models for the study of RNA genome mutations and
evolution. De la Torre had successfully established
persistent FMDV C infection by growth of BHK-21(c-13)
that survived standard cytolytic infection until massive
cell detachment .
The lysosomotropic weak bases, which diffuse into
acidic endosomes and raise the pH of endocytic
organelles, prevent the required low pH-dependent proteins
conformational changes leading to virus genome release.
As expected for a virus with an endosomal entry
pathway (such as FMDV), ammonium chloride neutralized
the acidic endolysosome compartments, blocking an
early step in virus infection. Previous studies have
confirmed that ammonium chloride can reduce the yield of
reovirus after infection and help establish persistent
infection rapidly in mouse L cells . So far as we
know, ammonium chloride has not been used for
selection and establishment of FMDV persistence previously
except studies in our lab. The influences of ammonium
chloride on the growth of FMDV were investigated first
to analyze the feasibility of establishing viral persistence
with the help of weak bases. The results in Figure 1
demonstrated that the presence of ammonium chloride
(10-40 mM) can reduce the yield of virus replication at
all time points detected.
A newly developed single-cell qRT-PCR  was used
in our assay to seek the optimal concentration of
ammonium chloride used in selection of persistent
infection with FMDV serotype O. The viral RNA
positive rate and the number of cells survived determine the
probability of selection of persistent infection. In
conclusion, approximately 0.015% (150/106) of the cells may be
virus persistent cell clones treated with 20 mM
ammonium chloride, which is 100-fold larger than that of cells
in the absence of ammonium chloride (0.00013%). The
results explained exactly why the persistence of FMDV
O (Akesu/58/2002) was not established merely by
growth of BHK-21 cells survived. The effect of the
multiplicity of infection on the establishment of persistent
infections with poliovirus in Hep-2 cell cultures had
been previously shown that low multiplicity of infection
favored establishment of persistent infection . Based
on these results, a very low multiplicity of 0.01 PFU/cell
was used for infection to establish FMDV persistence in
Evidence that BHK-Op cells were persistently infected
with FMDV includes (1): Detection of FMDV genome
RNAs by qRT-PCR in different passages of BHK-Op
cells. (2): Detection of FMDV specific proteins 3D and
3CD. The 3D and 3CD proteins were detected positive
in BHK-Op48 cells and the amounts of the viral
proteins were less than that in acutely infected cells. (3):
Observation of intact virions. The FMDV particles were
successfully observed in BHK-Op48 cells under the
transmission electron microscope.
In order to facilitate establishment of persistent
infection, ammonium chloride may act primarily on the host
cell or on the virus itself. To make it clear, we explored
the effects of ammonium chloride on both persistent
cells and viruses. As shown in Figure 8, infections of wt
viruses and persistent viruses were both sensitive to
ammonium chloride treatment. On the other hand,
results of MTT assays showed that the survival
probabilities of BHK-Op cells were much higher than that of
BHK-21 cells maintained in 20 mM ammonium
chloride. In other words, the persistent cells acquired
resistance to ammonium chloride. Interestingly, after
ammonium chloride treatment virus-resistant cells and
virus-sensitive cells were also selected and obtained. In
conclusion, ammonium chloride exerted its primary
effect on the host cell in the process of viral persistence
establishment. It is easy to explain the emergence of
virus-resistant cells because ammonium chloride may
result in phenotypic protection of certain
subpopulations of cells by preventing the virions from releasing,
which was consistent with previous studies [24,25].
However, virus-sensitive cells selected during the
establishment of persistence have never been reported yet,
which needs further and deeper research.
The virus suspensions of FMDV-Op8, FMDV-Op48
and FMDV wt were quantified by qRT-PCR to adjust
and equalize the amount of viruses used for infection in
the plaque assays and TCID50 assays. The fact that
infections of both FMDV-Op viruses resulted in no
plaques in BHK-21 cells while FMDV wt infection formed
many clear and visible plaques indicates that the
infectivity of FMDV-Op may be influenced and impaired in
the process of viral persistence. However, the result of
TCID50 assays implies that the virulence of FMDV-Op
viruses was enhanced compared to that of wild type
viruses. Therefore, virus internalization assays were
designed and carried out to investigate whether the
bindings of viruses to cell surfaces were blocked and the
possible reasons for vanishing plaques. After a 1 h (2 h)
absorption period, viral genome RNAs were detected in
cells infected with FMDV-Op8, FMDV-Op48 and
FMDV wt respectively, demonstrating that all the three
viruses could bind to cell surfaces and access to the
host cells. The amounts of viral RNAs in FMDV wt
infected cells were quantified more than 10-fold of that
in FMDV-Op infected cells at 4 h.p.i. Therefore, it
appears that FMDV-wt went through a natural
infectious cycle in the host cells with active viral proteins
transcription and genome replication. Meanwhile, the
life cycles of FMDV-Ops may be retarded and most of
the viral particles may lose the ability to uncoat and
retain in the endosomes, which is likely to be one of the
reasons for lost plaques. Furthermore, it has been
proved that ammonium chloride inhibits the required
low pH-dependent proteins conformational changes,
and blocks viral penetration of the cytoplasm rather
than inhibiting viral entry by blocking uptake at the cell
surface [15,18,30], which helps to clarify the function
and mechanism of ammonium chloride treatment.
Protein sequence analysis of wt viruses and persistent
viruses revealed that all the amine acid mutations were
situated in functional proteins rather than capsid
proteins. Based on the genome comparisons of 103 isolates
of FMDV representing all seven serotypes , 6 out of
15 mutations were the mutations of invariant amino
acids (1 in 2C protein, 1 in 3A protein, 3 in 3C protein
and 1 in 3D protein.) which may have some effect on the
functions of the viral proteins. Viral 3C protein is related
to the trypsin family of serine proteases and cleaves most
of the proteins from polyproteins. Mutations in the
conservative regions of 3C protein may result in the decline
of activity. Nayak and his coworkers found that the R95
and R97 residues of 3C protein were crucial for RNA
binding in virus replication . It was found that the
R95S and R97S mutant transcripts were non-infectious;
no plaques were observed at 8 h post electroporation.
However, the influences on virus life cycle of the amino
acid mutations are unclear and further investigations are
needed. Microarray experiments are underway to attempt
to search host cell proteins or factors pertaining to
FMDV persistence in BHK-21 cells.
In summary, persistent infection with FMDV O (Akesu/
58/2002) was established successfully with the aid of
ammonium chloride which can not be selected and
established merely by growth of BHK-21 cells survived
cytolytic infection. We found there were some unique
features of the persistent cells. First, the virions in
persistent cells were observed located mainly in endosomes
scattered in the cytoplasm. Secondly, infection of the
persistent viruses can not form plaques in host cells but the
virulence of FMDV-Ops was enhanced compared to that
of wild type viruses. Thirdly, there were no mutations of
amino acid in structural proteins which is not in line
with the previous studies . Moreover, the virus
internalization assays confirmed that the FMDV-Ops can
bind to cell surfaces after 1 h absorption. We believe that
this newly established persistent cell culture can provide
a system for elucidating mechanisms of viral persistence
in vivo. Besides, in vitro persistence is also a useful tool
for investigating virus and cell evolution and facilitating
the study of virus-receptor interactions, which can be
served to define genetic determinants of virulence and to
identify viral and host cell determinants involved in the
establishment of persistent infection. Moreover, the
establishment of persistent infections in cells in vitro
under the treatment of ammonium chloride may have
important implications for the use of lysosomotropic
drugs in vivo.
Cell and virus
BHK-21 cell is a clone of cells provided by China Center
for Type Culture Collection (CCTCC) where this study
was conducted. The virus strain of serotype O FMDV
(Akesu/58/2002) used in the present study was derived
from the Lanzhou Veterinary Research Institute, Chinese
Academy of Agriculture Sciences, which was cloned by
three successive isolations of plaques formed on the
BHK21 cells. BHK-21 cells were cultured in Minimum
Essential Medium (MEM, Life Technologies, Carlsbad, U.S.A.)
supplemented with 10% heat-inactivated fetal bovine
serum (FBS, Life Technologies, Carlsbad, U.S.A.) and
penicillin (100 units/ml)-streptomycin (0.1 mg/ml) at 37°C
with 5% CO2 and used to propagate virus stocks and
measure virus titers in plaque assays.
liquid samples were homogenized in 750 μl Trizol LS
reagent (Life Technologies, Carlsbad, U.S.A.). Total RNA
was extracted according to the manufacturer’s
One-step quantitative RT-PCR (qRT-PCR) assay for
detection of viral genome RNAs in acutely infected
qRT-PCR was performed using the Platinum®
Quantitative RT-PCR ThermoScript™ one-step Mastermix
Reagents Kit (Life Technologies, Carlsbad, U.S.A.). The
PCR primers and probe (listed in Table 2) for detecting
FMDV RNA located within the viral 3D genes encoding
RNA-dependent RNA polymerase, which are based on
nucleotide sequencing of serotype O (Akesu/58/2002,
GenBank accession no. AF511039; ). The qRT-PCR
was performed in a final volume of 50 μl consisting of
3 mM MgSO4, 0.2 mM of each dNTP, 1 μl
Script™ Plus/Platinum® Taq Enzyme Mix, 40 U RNase
inhibitor, 300 nM probe, 500 nM forward primer and
reverse primer, 5 μg bovine serum albumin (New
England Bio Labs, Beverly, U.S.A.) and 10 μl RNA
samples or standard RNA samples. The standard RNA
templates were prepared as described elsewhere . The
RT-PCR was carried out on Bio-Rad CFX96 real-time
PCR detection system (Bio-Rad Laboratories, Berkeley,
U.S.A.). Based on the manufacturer’s protocol, cDNA
was synthesized at 50°C for 30 min, and the PCR profile
was 95°C for 5 min, followed by 40 cycles of 94°C for 30
s and 60°C for 90 s. All the cell samples were tested in
triplicate. Data analyses were carried out using Bio-Rad
Establishment of persistent infection
Virus infections and treatment of infected cells with
BHK-21 cells in 6-well plates (or 12-well plates) were
infected with FMDV at a multiplicity of infection (m.o.i.)
of 0.01 PFU/cell. After 1 h of absorption at 37°C, cells
were washed for 1 min with 0.1 M phosphate buffer
(pH 6.0) to inactivate unabsorbed virions, and washed
again extensively with MEM. The infection was allowed
to proceed in MEM (5% FBS) supplemented without or
with 10, 15, 20, 30, 40 mM ammonium chloride for 48 h,
respectively. After removing the mediums with
ammonium chloride, infected cells were washed extensively
with MEM and then kept in fresh MEM supplemented
with 10% FBS for 24 h followed by single-cell clone
BHK-21 cells were centrifuged and homogenized with
400 μl Trizol reagent (Life Technologies, Carlsbad, U.S.A.)
in 1.5 ml Eppendorf polypropylene tubes while 250 μl
One-step single cell qRT-PCR assay
Single cells were isolated and lysed as described previously
. Single cell qRT-PCR was performed using the same
protocol described above. The PCR profile was 95°C for
Table 2 Synthetic oligonucleotides used for qRT-PCR
5 min, followed by 45 cycles of 94°C for 30 s and 60°C for
90 s. Data analyses were carried out using Bio-Rad CFX
Selection of cell clones infected persistently with FMDV
After 48 h treatment of ammonium chloride and 24 h
maintenance in fresh MEM, infected cells were rinsed
with PBS three times and then trypsinized at 37°C for
5 min. 9 ml 10% FBS MEM was then added to end
trypsinization. Single cells were isolated using a
micromanipulator (Narishige, Tokyo, Japan) and put onto a 96-well
plate (one cell per well) containing fresh MEM with 10%
FBS . The isolated single cells in separate wells were
allowed to form a monolayer, passaged and frozen as
usual which have been described previously .
A total of 5 × 106 BHK-21 cells, BHK-Op48 cells and
acutely infected BHK-21 cells (48 h.p.i.) were centrifuged
and washed with PBS (0.1 M) twice, respectively.
Harvested cells were fixed by 2.5% glutaraldehyde at 4°C
overnight. Samples were postfixed in 1% osmium
tetroxide for 2 h at room temperature, rinsed, and then
dehydrated in an up-graded ethanol series (30%, 50%, 70%,
80%, 90%, 95%, and 100%) and embedded in epoxy resin,
and ultrathin sections were double stained in uranyl
acetate and lead citrate. The ultrathinsections (60-80 nm)
were observed under a FEI TECNAI G2 transmission
electron microscope (FEI, U.S.A.).
Viral specific proteins, 3D (viral RNA dependent RNA
polymerase) and 3CD were identified by western blot
following the procedures: proteins extracted from
persistently and acutely infected BHK-21 cells by RIPA buffer
(20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5% NP-40,
0.5 mM EDTA, 0.5 mM PMSF, 0.5% protease inhibitor
cocktail [Roche]) were subjected to 12% sodium dodecyl
sulphate (SDS)-polyacrylamide gel, electrotransferred
onto a polyvinylidene fluoride (PVDF) membrane,
blocked with 5% nonfat milk in PBS, and reacted with
rabbit anti-FMDV 3D serum as primary antibodies.
Alkaline phosphatase (ALP)-conjugated goat anti-rabbit
immunoglobulin G antibody (Sigma, U.S.A.) was used as
the secondary antibody. After washing with TBS three
times, membrane-bound antibodies were detected with
Nitro Blue etrazolium/5-bromo-4-chloroindol-2-yl
Quantitation of FMDV positive- and negative-stranded
RNAs by duplex qRT-PCR
The duplex qRT-PCR for simultaneous detection of
FMDV positive-stranded RNAs and negative-stranded
RNAs has been developed and evaluated in our lab .
On the basis of the characteristics of FMDV genome, 2B
primers and probe were used for detection of
positivestranded RNAs while 3D primers-probe mentioned above
were designed for quantitation of negative-stranded RNAs
aiming at synthesis of different DNA fragments (or
nonoverlapped fragments) in the reverse transcription step.
The sequences of the primers and probes used in the
duplex qRT-PCR were shown in Table 2.
Reverse transcription was performed in a 20 μl reaction
mix containing 0.5 μl Transcriptor Reverse Transcriptase
(20 U), 4 μl 5 × Transcriptor RT Reaction Buffer (Roche
Diagnostics, Berlin, Germany), 0.5 μl RNase inhibitor
(20 U, TAKARA BIO, Shiga, Japan), 2 μl 10 mM dNTP
mix, 2 μl 25 μM 2B RT primer and 2 μl 25 μM 3D FP
primer, 4 μl DEPC water and 5 μl standard templates or
samples. The initial denaturation at 65°C for 5 min was
done with RNA samples and primers followed by snap
cooling on ice. Then, after adding enzyme, buffer and
dNTPs, the cDNA synthesis was carried out at 55°C for
30 min followed by heating at 85°C for 5 min to
inactivate transcriptase. cDNA products were cooled on ice
and stored at -30°C until use.
The optimum PCR reaction mixture contained 5 μl of
each 10-fold dilution of the 2B and 3D standard cDNAs
used to generate standard curves or 5 μl cDNA of cell
samples, 5 μl of 10 × LA PCR Buffer||, 500 nM each of
forward and reverse primers for 2B and 3D, 200 nM 3D
probe, 400 nM 2B probe, 400 nM of each dNTP and
2.5 U LA Taq. Distilled water was added to a total
volume of 50 μl. Amplification and detection were
performed with a Bio-Rad CFX96 real-time PCR detection
system under the following conditions: 95°C for 3.5 min,
followed by 40 cycles of 94°C for 30 s and 60°C for 90 s.
All the cell samples were tested in triplicate.
The house-keeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was examined and quantified as
an internal control for FMDV quantitation. Specific
primers and probe (see in Table 2) for GAPDH (GenBank
accession no. DQ403055) were designed using Primer
Express 2.0 (Applied Biosystems, Foster City, U.S.A.).
GAPDH mRNAs were detected with both one-step and
routine two-step qRT-PCRs using the same protocols and
conditions depicted above.
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), is reduced to purple
formazan in living cells. MTT assays were used to
determine the viability of BHK-21 cells and persistent
BHKOp cells treated with 0 mM (negative control) or 20 mM
ammonium chloride. Approximately 5000 cells (BHK-21
cells, BHK-Op8 cells and BHK-Op48 cells) were plated
separately into each well of 96-well plates in a 100 μl
volume and incubated overnight at 37°C with 5% CO2,
and then the supernatants were replaced by 100 μl 5%
FBS MEM containing 0 mM or 20 mM ammonium
chloride. Cell viability was measured at three different
time points: 24 h, 48 h maintenance in ammonium
chloride and 48 h maintenance in ammonium chloride then in
fresh 10% FBS MEM for 24 h. 20 μl 5 mg/ml MTT
(Sigma, St. Louis, U.S.A.) were added to each well and
cells were incubated in the culture hood for 4 h. After
that, the supernatants were removed and 100 μl DMSO
were added to dissolve the MTT (formazan). Cell viability
was determined with a microplate reader at 490 nm. Each
experiment was performed in sixteen replicate wells for
each sample and was repeated once.
Plaque assays and TCID50 assays
Virus was titered by standard plaque assay  with
some modifications. Briefly, confluent monolayer of
BHK-21 cells in 6-well plates as well as serial 10-fold
dilutions of wild type viruses and persistent viruses with
equal viral RNA copies in MEM were prepared, 0.1 ml
virus suspension was added to each well in duplicate for
each dilution after removal of the medium and washing
thrice. Infected cultures were then incubated at 37°C for
60 min with gentle shaking every 15 min to assure
uniform adsorption. Following this, cell monolayers were
overlaid with 3 ml maintenance medium (2% FBS)
containing 0.8% purified agarose and incubated at 37°C in a
5% CO2 incubator until plaques formed. 3-4 days later,
plaques were stained using 1 ml 1% crystal violet in 10%
paraformaldehyde and recorded after washing the
monolayer with tap water gently. TCID50 assays were
carried out using the routine method described
The significance of the results was tested using the
unpaired t test and the one-way ANOVA analysis of
variance using commercially available software
(Graphpad Prism, San Diego, USA). A P value less than 0.05
was considered significant. All values reported in the
text and figures were expressed as means ± standard
error of mean.
HX and LY conceived the study and designed the experiments. HX and FH
carried out the experimental work. HX, LY and ZCY wrote the paper. All
authors have read and approved the final manuscript.
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