Cultured early goat embryos and cells are susceptible to infection with caprine encephalitis virus.
ABSTRACT Zona-pellucida-free embryos at 8-16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days. After 5 weeks, multinucleated giant cells typical of CAEV infection were observed in indicator GSM cell monolayers. In the acellular medium, the early embryonic cells produced at least 10(3.25) TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEV and that infection with this virus is productive.
- SourceAvailable from: Berhanu Tameru[Show abstract] [Hide abstract]
ABSTRACT: Deriving horse oocytes in the USA is hampered by the lack of abattoirs processing horse carcasses which could provide abundant quantities of ovaries from slaughtered mares. Therefore, several cloning industries in the USA are attempting to import cloned horse embryos from Canada. Like any agricultural commodity, cloned embryos pose a risk of introduction of exotic animal diseases into the importing country. Under such circumstances, risk assessment could provide an objective, transparent, and internationally accepted means for evaluating the risk. This quantitative risk assessment (QRA) was initiated to determine the risk of introduction of Equine infectious anemia virus (EIAV) into the USA via cloned horse embryos imported from Canada. In assessing the risk, a structured knowledge base regarding cloning in relation to Equine infectious anemia (EIA) was first developed. Based on the knowledge base, a scenario tree was developed to determine conditions (with mathematical probabilities) that could lead to the introduction and maintenance of EIAV along the cloning pathway. Parameters for the occurrence of the event at each node were estimated using published literature. Using @Risk software and setting Monte Carlo simulation at 50,000 iterations, the probability of importing an EIAV-infected cloned horse embryo was 1.8 × 10(-9) (R = 1.5 × 10(-12) to 2.9 × 10(-8)). Taking into account the current protocol for equine cloning and assuming the yield of 5 to 30 clones per year, the possible number of EIAV-infected cloned horse embryos ranged from 2.0 × 10(-10) to 9.1 × 10(-5) (Mean = 1.4×10(-6)) per year. Consequently, it would take up to 1.5 × 10(7) (R = 1.6 × 10(4) to 5.1 × 10(10)) years for EIAV to be introduced into the USA. Based on the knowledge base and our critical pathway analysis, the biological plausibility of introducing EIAV into USA via cloned horse embryos imported from Canada is extremely low.Theriogenology 09/2011; 77(2):445-58. · 2.08 Impact Factor
- Revista Espanola De Cardiologia - REV ESPAN CARDIOL. 01/2011; 64(9):838-839.
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ABSTRACT: The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.Theriogenology 04/2008; 69(4):408-15. · 2.08 Impact Factor
Cultured early goat embryos and cells are susceptible to
infection with caprine encephalitis virus
M.Z. Ali Al Ahmada, F. Fienia,⁎, F. Guiguenb, M. Larrata,
J.L. Pellerina, C. Rouxa, Y. Cheblouneb,c
aDepartment of Research into the Health Risk and Biotechnology of Reproduction ENVN/DGER,
National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France
bUMR 754 INRA/ENVL/UCBL, Lyon Cedex 07, France
cMMD Lab, KUMC, 3901 Rainbow Blvd, 66160, Kansas City, KS, USA
Received 24 February 2006; returned to author for revision 3 May 2006; accepted 6 June 2006
Available online 21 July 2006
Zona-pellucida-free embryos at 8–16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-
pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and
cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days.
After 5 weeks, multinucleated giant cells typical of CAEVinfection were observed in indicator GSM cell monolayers. In the acellular medium,
the early embryonic cells produced at least 103.25TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions
within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected.
All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEVand that infection with this
virus is productive.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Embryo cells; CAEV; Replication; Tropism; Cytopathic effect
Lentiviruses are small, enveloped viruses with positive,
single-strand RNA, classified in the Lentivirus genus of the
Retroviridae family. They cause persistent infection, which
induces chronic degenerative disease in infected hosts
following a prolonged incubation period (Haase, 1986; Joag
et al., 1996). Other viruses of the Lentivirus genus include
maedi-visna virus (MVV) or ovine progressive pneumonia
virus (OPPV), equine infectious anemia virus (EIAV), feline
immunodeficiency virus (FIV), bovine immunodeficiency
virus (BIV), simian immunodeficiency viruses (SIV), human
immunodeficiency viruses (HIV) and caprine arthritis-ence-
phalitis virus (CAEV) (Evermann, 1990).
CAEV, like all lentiviruses, is an enveloped particle with a
diameter of 80–100 nm and a dense conical core, which
contains two copies of single-stranded genomic RNA and
various viral proteins (Gonda et al., 1986; Gelderblom, 1991).
CAEV was first isolated in 1980 from synovial membranes of
adult goats with arthritis and the brains of kids with encephalitis
(Crawford et al., 1980; Narayan et al., 1980). Typical clinical
symptoms of CAEV infection include leuco-encephalomyelitis
in young kids (2 to 6 months) (Cork et al., 1974; Narayan et al.,
1980) and arthritis and mastitis in adult goats (Crawford and
Adams, 1981). Unlike MVV-infected sheep that develop severe
interstitial pneumonia, severe lung disease is rare in CAEV-
infected goats (Crawford and Adams, 1981; East, 1996).
The high prevalence of CAEVinfection is a major concern in
many regions of the world, particularly in industrialized
countries (Dawson et al., 1983; Ellis et al., 1983; Belanger
and Laboeuf, 1990; Kreig and Peterhans, 1990; Perk, 1990;
Hanel, 1991; Rowe et al., 1992). However, only 10% to 25% of
Virology 353 (2006) 307–315
⁎Corresponding author. Fax: +1 33 2 40687748.
E-mail address: email@example.com (F. Fieni).
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
infected goats develop clinical signs of the disease (Crawford
and Adams, 1981; Vitu and Russo, 1988).
Cells from the monocyte–macrophage lineage are the
principal target of CAEV in vivo. The virus only replicates
following the differentiation of monocytes into macrophages
(Narayan et al., 1982, 1983). However, viral transcripts have
been detected in epithelial tissues from various organs such as
the kidney and gastrointestinal tract (Zink et al., 1990).
Epithelial cells in goat's milk have also demonstrated suscept-
ibility to CAEV infection both in vitro and ex vivo (Mselli-
Lakhal et al., 1999). More recently, CAEV provirus has been
detected in tissues of the genital tract: uterus, oviduct and
ovaries. Granulosa cells (Lamara et al., 2001) and epithelial
cells of the oviduct (Lamara et al., 2002a) have been shown to
be susceptible to CAEV infection, in vitro.
The mainroute for CAEV transmissionhas been shown to be
vertical from infected dams to newborn or young kids following
the ingestion of infected cells in the colostrum and milk.
However, other routes of infection have also been described.
Adams et al. (1983) reported seroconversion in 2 out of 32 kids
delivered by cesarean section and in 1 of 10 kids by natural
vaginal delivery, all of which had been deprived of colostrum
from their infected mothers. This indicated the possibility of
intra-uterine infection and prenatal vertical transmission. Other
reports have confirmed this in utero transmission with ovine
lentivirus infection from infected ewes to fetal lambs (Cutlip et
al., 1981; Brodie et al., 1994), and in addition, in some cases,
uterine disease was attributed to CAEV infection (Ali, 1987).
More recently, CAEV-infected cells have been detected in
flushing media from infected goats used as donor of embryo
(Fieni et al., 2002). In vitro studies have shown that the non-
deteriorated zona pellucida was a strong barrier that protects the
caprine embryo from CAEV infection but embryos lacking
zona pellucida when incubated with CAEV and washed
extensively, they could transmit the infection to the permissive
indicator GSM cells (Lamara et al., 2002b). However, it has not
been determined whether early embryo caprine blastomeres are
susceptible to CAEV infection and whether these cells are
viable or not following this infection.
In this study, our aim was to determine whether early embryo
cells of caprine blastocysts deprived from their zona pellucida
are susceptible to CAEV infection and whether these cells
support a productive replication of this virus. Using RT-PCR to
detect the viral genome in the culture medium, PCR to detect the
provirus in cellular DNA and immunocytochemistry to detect
the expression of the major p28 capsid protein, our results
demonstrate that early embryo blastomeres of goats without
zona pellucida are susceptible to infection with CAEV-pBSCA
and these cells support a productive replication of the virus.
This study was designed to clearly determine whether early
goat embryo cells can be infected with CAEV, whether they
support the replication of CAEV and finally whether they
produce a detectable titer of infectious cytopathic CAEV. PCR
and RT-PCR were considered as being positive when a specific
512-bp band was generated of a similar size to that observed in
the positive control; no specific bands were detected in samples
from the negative controls (Fig. 1).
Viral assay of culture medium of insert co-cultures and
At day 4 post-infection of GSM/COEC cells, just prior to
co-culture with early goat embryos, the supernatant fluid in
the monolayer compartment contained 105.5
while that in the top of the insert contained 104.75
TCID50/ml as determined by virus titration on GSM cells
as described in Materials and methods. After 6 days of co-
culture of early goat embryos in inserts on CAEV-infected
cell monolayers, embryos were harvested and washed 10
times over by successive passages in fresh medium in tissue
culture wells. Culture medium of the co-cultures and each of
the 10 successive baths were examined by RT-PCR to detect
CAEV RNA of free cell particles. As shown in Table 1, the
culture medium of the co-culture was found to be positive,
as expected. Interestingly, only media from the 4 first baths
contained CAEV particles, while subsequent baths were
consistently negative. Culture medium and all washing fluids
from the control groups and embryos co-cultured with non-
infected monolayers were consistently found to be negative.
These results clearly show that for early goat embryos co-
cultured in infectious conditions the five first washes were
Fig. 1. Representative nested PCR amplification of provirus DNA. One microgram of DNA was used from each sample, as described in the Materials and methods
section, to perform nested PCR using CAEV-gag-specific and β-actin-specific sets of oligonucleotide primers. Following nested PCR reactions, 10 μl of each PCR
product was separated on 1.5% agarose gel and the bands visualized by staining with ethidium bromide. Oligonucleotide primers were used to amplify the 512-bp
CAEV gagand 393-bp-actin fragmentsshownwith the arrows. M: 50-bpDNA Ladder usedas a molecular weightstandard.Lanes 1 and 2: DNA isolated from CAEV-
infected GSM cells. Lanes 3 and 4: DNA isolated from GSM control cells (negative). Lanes 5 to 8: DNA isolated from infected embryo cell monolayers. Lanes 9 to 11:
DNA isolated from control embryo cell monolayers (negative). C+: positive control (CAEV proviral DNA). C−: negative control (distilled water).
308M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
sufficient to remove any residual viral particles from the
CAEV transmission from inoculated early goat embryos to
To investigate whether in vitro CAEV-infected early goat
embryos are capable of transmitting CAEV to target cells, co-
cultured embryos washed 10 times over were transferred to a
co-culture in an insert (batch 1) or in direct contact (batch 2)
with fresh non-infected GSM cells. After 6 h of co-culture,
CAEV RNA genome was detected by RT-PCR in the medium
of co-cultured embryos from two batches in the CAEV group
but in none of those from the control group. Embryos were
removed from the co-culture after 6 h and the GSM cells
subcultured for 5 weeks. Multinucleated giant cells typical of
CAEV infection were observed in GSM cell monolayers that
had been co-cultured with CAEV-infected embryos, but not in
those co-cultured with non-infected embryos (Fig. 2). CAEV
provirus genome was also repeatedly detected in DNA samples
from GSM cells co-cultured with CAEV-infected embryos but
never with the non-infected control embryos (Table 2).
Fig. 2. Typical CAEV-induced cytopathic effects in GSM cells following co-culture with infected embryos in an insert or in direct contact. Non-infected GSM cells (4)
and co-culture of GSM cells with CAEV-infected blastomeres (1) at 50× magnification (photo 4: scale bar=160 μm and photo 1: scale bar=180 μm). Typical CPE
were shown at 100× (2) and 200× (3) magnification (photo 2: scale bar=90 μm and photo 3: scale bar=45 μm).
Analysis of CAEV transmission from inoculated goat embryos to GSM target
Goat synovial membrane (GSM) cells used as viral indicator cell lines were co-
cultured for 6 h with infected embryos and non-infected embryos in an insert or
directly in contact. GSM indicator cells were subcultured following trypsiniza-
tion each 6 days and maintained in culture for 5 weeks. Presence of cytopathic
effects (CPE) was detected by phase microscopy, and the 512-bp band specific
to CAEV gag was detected following migration of the PCR products on 1.5%
agarose gel. Positive and negative results for the six repetitions of the
experimental procedure were checked and reported in the table.
RT-PCR analysis of culture medium of co-cultures and embryo washing fluids
Culture medium of early goat embryos co-cultured with non-infected or CAEV-
infectedCOEC/GSMcell monolayerswasharvestedatday6 postco-cultureand
examined using RT-PCR. In parallel, harvested embryos were successively
passed through ten baths and all washing fluids examined individually by RT-
PCR. The 512-bp band specific to CAEV gag was detected following migration
of the RT-PCR products on 1.5% agarose gel. RT-PCR positive and negative
samples for the six repetitions of the experimental procedure were scored and
reported in the table.
309M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
Altogether, these data clearly demonstrate that early goat
embryos that have been exposed to CAEV are capable of
transmitting this virus, via particles produced de novo, to
susceptible target cells.
Titer determination of CAEV produced by infected early goat
To determine the infectious cytopathic titer of CAEV
produced and released in the culture medium of early goats
embryos infected in vitro, the supernatant fluid of cultured
CAEV-infected embryos was harvested 24 h after washing and
culture. The supernatant was cleared by filtration and various
dilutions used to inoculate fresh GSM cell monolayers. Using
the Reed method, CAEV production by infected early goat
embryos was found to be between 103.25(minimum) and 104.5
CAEV replication in cultured early goat embryo cell
Embryos were subjected to the action of trypsin, and the
resulting dissociated embryo cells were cultured as monolayers.
At days 4 and 8 of culture, samples were examined by PCR and
RT-PCR. DNA isolated from cell cultures derived from CAEV-
infected embryos showed the 512-bp product specific to CAEV
gag. No CAEV-specific PCR product was amplified with DNA
isolated from cultured cells derived from non-infected embryos.
The 512-bp specific gag product was also amplified following
RT-PCR analysis of samples of supernatant fluid taken from
cultured CAEV-infected embryo cells, but not from that of non-
infected cultured embryo cells (Table 3). Immunocytochemistry
analysis of cultured early goat embryo cells, using an anti-p28-
gag-specific monoclonal antibody, confirmed the expression of
viral protein in these cells (Fig. 3).
These data clearly demonstrate that CAEV-infected early
goat embryo cells are productively infected and CAEV
replication is supported by these cells.
The aim of this study was to examine the susceptibility of
early goat embryos and derived cell cultures to CAEVinfection.
We have previously reported that the intact zona pellucida of
goat embryos efficiently protects them from CAEV infection,
whereas early goat embryos without zona pellucida were
Analysis of CAEV productive replication in cultured early goat embryo cell
Infected embryo cells Non-infected embryo cells
CAEV proviral DNA
Infected and non-infected embryos were harvested and treated with trypsin to
dissociate the cells that were cultured as monolayers over 4 to 8 days. Cells from
the monolayers were harvested following treatment with trypsin; a portion of
these cells was used for DNA and RNA isolation for detection of provirus using
PCR and of viral RNA by RT-PCR, and a second portion was used for
examinationby immunocytochemistry to detect the expressionof the major viral
p28 gag protein. Positive and negative results for the six repetitions of the
experimental procedure were checked and reported in the table.
Fig. 3. Immunocytochemistry analysis of CAEV major capsid: p28 gag expression in cultured early goat embryo cells. Early goat embryo cultured cells in slide
chambers, derivedfromCAEV-infectedembryos(3)or non-infected embryos(4),werestainedby immunocytochemistryusinga monoclonal antibodydirectedagainst
the major gag p28 capsid protein. Non-infected (2) and CAEV-infected (1) GSM cell monolayers were used as controls. The dark brown staining (in panels 1 and 3)
corresponds to viral protein expression (photo 1: scale bar=160 μm, photo 2: scale bar=170 μm, photo 3 and photo 4: scale bar=70 μm).
310 M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
capable of transmitting CAEV to susceptible target cells
following co-culture (Lamara et al., 2002b). However, the
data in this earlier study were not sufficient to determine
whether this viral transmission was generated from the initially
introduced, wash-resistant residual CAEV, or from CAEV
produced de novo by early goat embryos following productive
infection of their cells.
In this study, we developed co-culture systems either in
direct contact or with a membrane separation between COEC/
GSM cell monolayers and the embryos. This insert co-culture
system helped to exclude the contamination of cultured
embryos or embryo cell cultures with feeder COEC/GSM
cells from the monolayer. Interestingly, we found the viral titer
to be approximately 1 log lower in the medium on top of the
insert, compared to that in the lower compartment directly in
contact with the feeder CAEV-infected monolayer. This
difference could be explained by the reduced permeability of
the insert membrane following several days of culture. The
results reported in this work clearly demonstrate the different
stages of viral infection of early goat embryos with CAEV.
We demonstrated that CAEV can adhere to embryos and that
4–5 successive washings by serial passages in fresh medium
were sufficient to remove this virus. Secondly, embryos that
have been washed 10 times over were shown to be capable of
transmitting CAEV to susceptible GSM cells following co-
culture. Thirdly, viral genome and infectious particles were
detected in the culture medium of cultured embryos and embryo
cells. Furthermore, the proviral genome, viral genome and the
major viral p28 gag protein were detected in CAEV-infected
embryo cells. Finally, CAEV titers ranging from 103.25to 104.5
TCID50/ml were found to produce infected embryos following
24 h of culture.
These data demonstrate that not only are early goat
embryo blastomeres susceptible to CAEV infection but that
they support its replication to produce reasonable titers of virus
released into the extra-cellular compartment. These data
together with those published earlier (Lamara et al., 2002b)
help to conclude that the only barrier to prevent CAEVinfection
of goat blastomeres in nature is the presence of an intact zona
Interestingly, this ability of the ZP to protect blastomeres
against viral infection is not restricted to CAEV, it has also
been described for Bovine BHV-1 that was shown to be
capable of causing infection in early ZP-free goat embryos at
the 8-cell stage (Vanroose et al., 1997) and BTV at the morula
stage (Bowen et al., 1982). Similarly, early embryo cells were
shown to be susceptible to BTV infection (Bowen et al., 1982).
However, bovine, mouse and swine ZP-free early embryos
were shown to be resistant to bovine parvovirus (BPV),
cytomegalovirus (CMV) and pseudorabies virus (PRV),
respectively (Neighbour, 1978; Bowen, 1979; Bolin et al.,
1981). These data suggest a differential susceptibility of early
embryos to viral infection, which may be related to the
presence of: (1) functional receptors expressed at the surface of
these cells, (2) mature transcription factors that allow their
replication and (3) absence of repressors of virus replication.
Indeed, the entry of enveloped viruses is strictly regulated by
the presence of functional receptors expressed at the surface of
target cells. This restriction led us to choose early goat
embryos at the 8–16 cell stage, which are known to show
active gene expression in various mammalian species (Crosby
et al., 1988; Barnes and First, 1991; Kelk et al., 1994). The risk
of disrupting the zona pellucida is minimal in vivo in early
goat embryos, whereas it is increased during ex vivo and in
vitro manipulation for embryo transfer, which therefore
increases the risk of CAEV infection of early embryos. Several
studies have demonstrated that the infection of early embryos
by BVDV (Booth et al., 1998; Vanroose et al., 1998;
Stringfellow et al., 2000) or BHV-1 (Vanroose et al., 1997)
interferes with the in vitro development of bovine embryos.
However, this interference was found to be dependent on the
virulence of the BVDV strains. In our previous study, we did
not find any effects of CAEV infection in either ZP-intact or
ZP-free early goat embryos (Lamara et al., 2002b). In addition,
no significant difference was observed between CAEV-
infected and CAEV-negative goats in terms of the average
number of embryos produced and their development following
transfer to recipient goats. The possibility that CAEV infection
of early goat embryos does not alter their development
increases the risk of the emergence of endogenous CAEV
genomes that could be transmitted vertically through germ
cells. This type of transmission affects every cell of the
organism resulting in at least 1 provirus per cell, as observed
for the endogenous JSRV (jaagsiekte sheep retrovirus) and
ENTV (enzootic nasal tumor virus) oncoretroviruses of sheep
and goats. To date, no endogenous lentivirus genome has been
identified in any mammals following natural lentivirus
infections. This absence of endogenous lentiviruses could be
a result of the efficient ZP protection of embryo blastomeres
from CAEV infection. Our findings confirm and support the
IETS recommendation of ZP-intact embryo washings and
bring an additional restriction concerning the strict elimination
of any non-ZP-intact embryo for embryo transfer. This will
minimize the risk of the emergence of CAEV endogenous
genomes in animals resulting from embryo transfer in CAEV-
CAEV infection of early embryo cells
To examine the susceptibility of early goat embryo cells to
caprine arthritis-encephalitis virus (CAEV) infection and
replication in vitro, 185 zona-pellucida-free 8–16 cell
embryos, obtained from 30 donors, were used in this
experiment. Embryos were randomly divided into two groups:
two thirds (121) were used in the infected group and one third
(64) in the control group. Embryos from the infected group
were delicately laid on the membrane of tissue culture inserts
(Transwell, Corning 3450, Cambridge, MA) and then placed
onto a mixed 80% COEC (Caprine Oviduct Epithelial Cells)
and 20% GSM (Goat Synovial Membrane) monolayer cell
culture, which had been previously infected with CAEV-
pBSCA at a multiplicity of infection of 1 (MOI=1), in B2
311M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
medium (INRA, Ménézo, Paris France) supplemented with
10% fetal calf serum (FCS). Co-cultures were incubated at
38.5 °C, 5% CO2in a humidified atmosphere for 6 days. At 4
days post co-culture, a sample of supernatant fluid was
harvested, cleared and serial dilutions used to inoculate fresh
GSM cells for virus titer determination. Control group
embryos were subjected to similar conditions, except that
the monolayer of co-cultured cells was not infected with
After 6 days of co-culture, the control and infected
embryos were harvested and washed by successive passages
in ten separate wells containing 2 ml of MEM (Minimum
Medium Essence). The presence of CAEV was examined by
RT-PCR in insert culture medium and in the ten washing
Detection of CAEV infection in early goat embryo cells
To determine whether washed embryos release infectious
CAEV, a fraction aliquot (28 infected embryos) was
transferred for co-culture in an insert (batch 1) and a second
fraction aliquot (13 infected embryos) was directly co-
cultured in contact (batch 2) with GSM cell monolayers for
6 h. A sample of the culture medium was then taken for RT-
PCR analysis to identify whether CAEV was released or
excreted by the embryonic cells or to identify any free virus
liberated by the cells. Embryos were harvested for DNA
isolation for PCR analysis of CAEV provirus. Non-inoculated
and ZP-free embryos used as control (respectively 13 and 7
embryos) were also cultured under the same conditions and
were used as a negative control. GSM cells were maintained
in culture for 5 weeks. Every 24 h, half of the medium was
replaced. Every 6 days, GSM cells were subcultured
following trypsinization. Half of the cells were used to detect
CAEV DNA provirus by PCR, and the second half were used
to process a new culture. When DNA provirus was detected,
GSM cell cultures were formalin-fixed (10%), Giemsa-stained
and examined for the presence of cytopathic effects (CPE) by
Detection of CAEV productive replication in early goat
embryos and cultured embryo cells
To examine virus production, CAEV-infected and non-
infected control embryos were washed 10 times and then
cultured for 24 h in a cell-free medium (0.5 ml of B2 medium
supplemented with 10% FCS and 2.5 μg ml−1Fungizone at
38.5 °C in 5% CO2in a humidified atmosphere). After 24 h, the
medium was cleared by filtration through a 0.22 μm membrane,
serially diluted and used to inoculate fresh GSM cell
monolayers for the detection of CPE and the determination of
viral titers. Embryos were then harvested and treated with
trypsin to dissociate the cells that were cultured as monolayers
over 4 to 8 days. Cells from the monolayers were harvested
following treatment with trypsin; a portion of these cells was
used for DNA and RNA isolation for detection of provirus using
PCR and of viral RNA by RT-PCR, and a second portion was
used for examination by immunocytochemistry to detect the
expression of the major viral p28 gag protein.
Materials and methods
Embryos, cells and viruses
Embryos at the 8–16 cell stage were collected surgically
from thirty healthy 1- to 4-year-old Saanen or French Alpine
goats that were taken from 5 reputable certified CAEV free
herds. These animals have been consistently seronegative
throughout their life. Before their use as donors, the goats
were tested individually using an agar gel immunodiffusion
test and PCR detection of CAEV provirus in white blood cells
and in cervical smears. Superovulation, estrus behaviour and
surgical embryo collection have been described previously
(Lamara et al., 2002b). In short, does were synchronized for
estrus using an 11-day treatment with intravaginal sponges
impregnated with 45 mg flurogestone acetate (Intervet,
Angers France) and the intramuscular (IM) injection of
125 μg of a prostaglandin analogue (Estrumate, Shering-
Plough Veterinaire, France) 48 h prior to sponge removal.
Superovulation was induced following IM treatment with
porcine (p) FSH (Liège University, Belgium) twice daily for 3
days, starting on day 9 of the progestogen treatment. A total
of 16 mg Armor pFSH was injected at decreasing incremental
doses (4-4-2-2-2-2). The FSH/LH ratio was decreased from 8/
1 (day 9) to 4/4 (day 10) and 4/10 (day 11) by the addition of
purified pLH (porcine luteinizing hormone, Liège University,
Belgium) to the treatment. These does were mated with a
CAEV seronegative Alpine buck of proven fertility. Four to
five days after estrus, a laparotomy was performed for the
collection of embryos. The does were anesthetized with
Zolétil 100(ND)(1 ml/kg−1: tiletamine+zolazepam; Virbac,
Nice, France) and were prepared for aseptic surgery. The
uterus and ovaries were exposed via ventral midline
laparotomy. Forty milliliters of modified phosphate-buffered
saline (PBS) (Eurobio, Paris, France) was used to flush the
embryos from the oviducts and into the uterine horns, where
embryos were recovered using a Foley catheter. The embryos
were collected into a small sterile bottle. Abdominal and skin
closure followed routine procedures. Embryos with a good
quality, intact zona pellucida (ZP) were selected. To remove
the ZP, embryos were placed into a 3-ml Petri dish containing
1% pre-incubated pronase (Protease, P 6911 Sigma, Paris
France) at 37 °C. After 60–90 s, the embryos were transferred
into a 2-ml Petri dish containing pre-incubated acidic
Tyrode's solution (pH 2.1) at 37 °C for 90–120 s. Finally,
embryos were washed three times with Minimum Essential
Medium (MEM, Gibco-BRL, Paris France) supplemented
with 10% fetal calf serum (FCS).
Caprine oviduct epithelial cells (COEC) were used to
support the embryos in vitro culture. Oviduct cells were
obtained surgically from CAEV seronegative goats. Fallopian
tubes were flushed with trypsin 0.25%+0.02% EDTA in BPS
without Ca2+and Mg2+. After 10 min of incubation at 37 °C,
oviductal mucosal tissue was extracted and transferred into
312M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
10 ml of MEM. Cells were dissociated following repetitive
gentle pipetting. After centrifugation (700×g, 10 min, 4 °C), the
cell pellet was resuspended in 10 ml MEM supplemented with
10% heat-inactivated (56 °C, 30 min) FCS. Cell suspensions
were transferred into tissue culture dishes (Nunc Polylabo, Paris
France) and incubated at 38.5 °C with 5% CO2in humidified
air. The medium was replaced after 48 h, and resulting
monolayers were used for co-culture with embryos.
Goat synovial membrane (GSM) cells were used as viral
indicator cell lines. They were obtained originally from
explanted carpal synovial membrane from a colostrum-deprived
newborn goat (Narayan et al., 1980). The cell lines were
expanded by cultivation in MEM+10% FCS and stored in
liquid nitrogen. GSM cells are highly susceptible to fusogenic
infection with CAEV.
Caprine arthritis encephalitis virus-pBSCA was produced
from pBSCA, a plasmid carrying the complete CAEV genome
of the CO strain as described previously (Mselli-Lakhal et al.,
1999). In short, pBSCA plasmid DNA was introduced into the
GSM cells by transfection, and culture medium containing
released virus particles was then harvested. Virus stocks were
titrated on GSM cells and macrophages at 106TCID50/ml of
Cell infection, virus identification and titration
To obtain a supportive medium for embryo culture and viral
infection, cell monolayers were seeded into six-well plates
(Costar 3516-Fisher, Elancourt, France) at a density of 105cells
(80% CEOC and 20% GSM) in 2 ml of MEM per well. When
the monolayers were sub-confluent, cells were inoculated with
CAEV-pBSCA at a multiplicity of infection of 1 (MOI=1) and
incubated at 37 °C, 5% CO2in a humidified atmosphere. Six
days post-infection, infected cells were dissociated by trypsin,
divided into two wells and cultured. ZP-free 8–16 cell embryos
were added to the culture when the CEOC-GSM monolayer was
To identify CAEV infection in the monolayers, cells were
fixed in formalin (10%), stained with May-Grünwald-Giemsa
solutions and examined for cytopathic effects (CPE), presenting
as multinucleated giant cells resulting from the fusion of several
infected and non-infected cells.
To determine the infectious titers of viruses produced, the
supernatants of infected cultures were harvested, cleared by
filtration through 0.22 μm membrane and serially diluted in
medium. Ten-fold dilutions were used to inoculate GSM cells in
24-well plates, and infected cells were maintained in culture for
6 days. Any CPE that developed in the monolayers was scored
and virus titers calculated using the Reed–Muench method
(Reed and Muench, 1938) and expressed as tissue culture
infectious doses (TCID50) per ml of supernatant.
To identify the specific CAEV p28 capsid protein, infected
and control embryo cells were grown to sub-confluence in 8-
chamber slides (Lab-Tek, Illkirch, France) then fixed in cold
acetone. Viral antigens were tagged by incubation for 1 h at
room temperature with mouse monoclonal antibodies directed
against CAEV p28 (VMRD, Pullman, WA, USA) diluted 1:500
in 1% BSA in PBS. After washing, the slides were incubated for
a further 30 min at room temperature with a 0.5% solution of
biotinylated goat–anti-mouse Ig purified antibody (Dako; kit
K0433, Glostrup, Denmark) in 1% BSA in PBS, rinsed, stained
in diaminobenzidine (Dako kit K0433) for 8 min and counter-
stained with hematoxylin before being mounted as described
previously (Mselli-Lakhal et al., 1999). Unrelated murine
antibodies of the same Ig class were used as negative controls,
and GSM-infected cells as positive controls.
PCR amplification of proviral DNA
Infected and non-infected embryo cells and infected and
freshly prepared GSM (105cells) were lysed as described
previously (Chebloune et al., 1996). Total DNA was purified
using a “QIAamp DNA kit” (Qiagen, Courtabœuf, France)
according to the manufacturer's instructions. CAEV proviral
DNA was examined using nested PCR (Guiguen et al., 2000).
CAEV gag sequences were amplified using primers GEX5
(5′-GAA GTG TTG CTG CGA GAG GTG TTG-3′) and
GEX3 (5′-TGG CTG ATC CAT GTT AGC TTG TGC-3′),
corresponding to bases 393–416 and the complement of bases
1268–1291 of CAEV-CO (Saltarelli et al., 1990). Samples of
10 μl of isolated DNA (containing 0,5 to 1 μg) were used as a
template for PCR amplification (94 °C–1 min, 46 °C–1.5 min
and 60 °C–2.5 min). Amplification was preceded by an initial
denaturation at 94 °C for 5 min and terminated with a final
extension at 60 °C for 15 min. Five microliters of the PCR
product of this reaction was used as a template for a second
round of amplifications using the internal primers GIN5 (5′-
GAT AGA GAC ATG GCG AGG CAA GT-3′) and GIN3 (5′-
GAG GCC ATG CTG CAT TGC TAC TGT-3′), located at
positions 524–546 and 1013–1036 in CAEV-CO. Sample
DNA integrity was checked by amplifying the β-actin gene
using primers based on the human sequence (Joag et al.,
1994). Amplified bands were visualized via ethidium bromide
staining after electrophoresis through 1.5% agarose gel. This
technique has been shown to be capable of detecting less than
10 infected cells in samples containing 106or 107cells
(Chebloune et al., 1996).
RT-PCR amplification of viral RNA
RT-PCR was used to detect the CAEV genome in embryo
washing fluids and culture media either from culture in the
insert or from monolayers. RNA was purified using a
“QIAamp ARN kit” (Qiagen, Courtabœuf, France) in accor-
dance with the manufacturer's instructions. For each sample,
5 μl (containing 10–100 ng) of the total RNA extracted was
used as a template for RT with 15 μl of mix solution
containing: 1 μl of a dNTP mixture (25 mM each of: dATP,
dGTP, dCTP, dTTP), 1 μl of Random primers for RT (Biolabs,
S1230S, Ozyne, France), 4 μl of 5× RT buffer (Kit M-MLV
Reverse-Transcriptase, Promega, 3681, Charbonniéres Les
313 M.Z. Ali Al Ahmad et al. / Virology 353 (2006) 307–315
Bains, France), 2 μl of RT (Kit M-MLV Reverse-Transcriptase,
Promega, 3681) and 7 μl RNase-free water. The mixture was
incubated at 37 °C for 30 min. The reaction was then stopped
following incubation at 95 °C for 5 min, and the samples were
stored at −80 °C for subsequent PCR analysis. The latter
consisted of nested PCR with two amplifications of the gag
gene (Leroux et al., 1997).
The authors would like to thank G. Chatagnon, Sylvie
Anezo and Hervé Guilloteau from the Department of
Research into the health risks and biotechnology of
reproduction, National Veterinary School of Nantes, France,
for their technical assistance. This research was supported by
l'Institut de l'Elevage, France. The authors would also like to
thank Mr. J.P. Sigwal and Dr. E. Manfredi who managed the
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