Molecular characterization of the Egyptian strain (Iman) of

BVDV and its RNA synthesis in cell culture

(Accepted: 20.08.2000)

Hussein A. Hussein
Department of Virology, Faculty of Veterinary Medicine, Cairo University,12211 Giza, Egypt

ABSTRACT

In this study, a nested polymerase chain reaction (PCR) that distinguishes between three
pestiviruses, bovine viral diarrhea virus (BVDV) type I, BVDV type II as well as border disease
virus (BDV), was applied to identify and characterize the Egyptian Iman strain of BVDV. A 826 bp
fragment of the genome located in the gp 48 region of BVD viral genome was reverse transcribed
and amplified using consensus oligonucleotide primers in a RT-PCR assay. The amplified product
was then subjected to second amplification cycles in the presence of type-specific primers (nested
PCR) which revealed products with characteristic sizes for each of three viruses (BVDV type I,
BVDV type II and BDV). Utilizing this assay, the Egyptian strain of BVDV (Iman) was identified as
BVDV type I. The virus (Iman strain) was propagated for 16 successive passages on MDBK cell
line to study its cytopathic behavior as well as its RNA synthesis. RT-PCR assay utilizing other
primers that are located in the 5’ untranslated (UTR) region of the BVDV genome was applied to
follow up the BVD viral RNA synthesis and release in cell free and cell associated harvests. Viral
RNA was detected in cell associated harvests starting 4 hours post inoculation of BVDV in MDBK
cells and at 8 hour in case of cell free harvests.

Pestivirus, Bovine viral diarrhea virus, polymerase chain reaction, genotyping,

diagnosis.

INTRODUCTION 1989; Bolin and Ridpath, 1992 and 1995;

Ridpath et al., 1994; Pellerin et al., 1994;

T
he family Flaviviridae consists of three
genera: Pestivirus, Flavivirus and
Hepacvirus (Pringle, 1999). The
Pestivirus genus is composed of three
economically important viruses that infect
ruminants, namely bovine viral diarrhea virus
(BVDV), border disease virus (BDV and the
classical swine fever virus (CSFV) (Collett,
1992; Moening and Plagemann, 1992,
Wengler. et al., 1995). Recently, an additional
fourth pestivirus species isolated from cattle
and sheep has been reported (Corapi et al.,
Becher et al., 1995; Tijssen et al., 1996).
Pestiviruses have single stranded, positive
sense RNA genome that are approximately
12.5 kb in length and contain one large open
reading frame (ORF) flanked between 5′ and
3′ highly conserved untranslated regions
(UTRs) (Collett et al., 1988). On the basis of
sequence and phylogenetic analysis of
common regions of different pestiviruses, at
least 4 genotypes have been reported including
classical BVDV strains: NADL, Osloss, SD1
and R27/27 (pestivirus type 1); CSFV strains
buffer (10X buffer contains 500 mM KCl, 100
mM Tris-HCl, pH 8.3, 25 mM MgCl2), 0.2
mM of each primer P1 and P2, 200 µM (each)
dATP, dCTP, dGTP and dTTP, 2.5 units of
Taq polymerase (GIBCO), was subjected to
one cycle 94 oC for 1 min at 30 amplification
cycles of 1 min at 94 oC, 1 min at 55 oC and 1
min at 72 oC and finally one 7 min cycle at 72
o
C. The amplification products were analysed
by electrophoresis on 1.5% agarose gels. The
size of the PCR products were confirmed by
visualization on an ethidium bromide-stained
agarose gel, along with a molecular weight
marker. The second round of amplification
was applied using P2 primer and a cocktail of
type specific primers (TS1, TS2 and TS3) in a
nested PCR assay. Briefly, 1 µl from the first
round amplification product (diluted 1/100 in
nuclease free water) was added to the same
PCR reaction mixture but containing 1 µM
each of P2, TS1, TS2, and TS3 primers. The
second PCR amplification consisted of 25
cycles of 94 oC for 1 min, 50 oC for 45 sec and
72 oC for 45 sec. PCR products were loaded
onto 2% agarose gels stained with ethidium
bromide along with a molecular weight marker
(PCR marker, Promega Corporation).
Electrophoresis was conducted for 45 min at
120 V and the gels were examined under
ultraviolet light and photographed on Polaroid
type 660 film.
One step growth curve of BVDV-Iman
strain using RT-PCR
MDBK cells were prepared in tissue
culture flasks and adjusted to be 4 x 105 cells /
tissue culture flask (p25). The cells were then
inoculated with BVDV (Iman strain) and all
the flasks including the controls were
incubated for 5 days at 37 oC with daily
recording of CPE. Cell free and cell
associated virus in inoculated flasks as well as
the control cells and thier supernatants were
harvested according to scheduled time at: 4 hr,
8 hr, 12 hr, 20 hr, 24 hr, 30 hr, 48 hr, 72 hr, 96
hr and 120 hr. To characterize RNA synthesis
of BVD viral genome in the cell associated
and cell-free harvests of the inoculated cell
culture, all harvests were subjected to RNA
extraction and cDNA synthesis as described
above. PCR amplification was utilized using a
pair of primers (UTR1 and UTR2) to amplify a
region (288 bp) located in the 5′ UTR region
of the BVDV genome. The PCR reaction
mixture was the same as mentioned above in
the first amplification round with substitution
of P1/P2 primers by UTR1 and UTR2 primers.
The PCR cycles consisted of one cycle at 94
o
C for 1 min, 34 cycles at 94 oC for 45 sec,
55oC for 1 min and 72 oC for 1 min and a 7-
min-cycle at 72 oC. PCR products were
analysed on 2% agarose gels stained with
ethidium bromide along with a molecular
wieght marker (PCR marker, Promega
Corporation). Electrophoresis was conducted
for 45 min at 120 V and the gels were
examined and photographed as mentioned
above.
RESULTS
CPE of BVDV Iman strain on cell culture
In this study, 16 passages of BVDV
(Iman strain) on MDBK cell line were applied
to study the CPE. The first sign of CPE,
characterized by early rounding and
granulation of the infected cells in scattered
areas of monolayer, were observed during the
first day post infection (PI). The CPE was
then increased vigorously till day 4 PI.
Intracytoplasmic granules coalesced to form
enlarged holes spanned by cellular filaments
and surrounded by dark irregular cells have
been observed by the third day PI. In day 4 PI,
vaculation and foamy appearance were
observed in 90% of the cells followed by
complete destruction and detachment of cells
in the early hours of the fifth day PI. Table (1)
 MDBK cells were negative for amplification
in the PCR-based genotyping assay utilized in
the present study.
1):Agarose gel analysis of the PCR
products amplified with the type-
specific primers (TS1, TS2 and TS3).
Lane M, represents the DNA ladder
fragments with sizes of 50, 150, 300,
500, 750 and 1000 bp. Lanes A and
Fig.(2):Agarose gel analysis of the PCR
B, the PCR products of approximately
223 bp (BVDV type I) of BVDV
reference strain (NADL) and local
BVDV (Iman strain), respectively.
Lane C, the PCR products of
approximately 448 bp (BVDV type II)
of A125 reference strain of BVDV.
Lane D, the amplification products
from the negative control cells (non
infected MDBK cells).
One step growth curve of BVDV-Iman
strain on the level of RNA synthesis
Studying the time of RNA synthesis
and sequential follow up of the virus particle
release from the inoculated cells had been
carried out in scheduled time table by
detection of the BVD viral RNA in a RT-PCR
assay utilizing a primer set located in the
highly conserved 5’ UTR region of the BVD
viral genome (Collett et al., 1988). The
primers were selected to amplify stretch of the
5′ UTR region of 288 bp as the sense and
antisense primers were located at 108 -128 nt
and 395-375 nt sequence of the genome of
NADL strain of BVDV, respectively. The
PCR employed in this study correctly
identified BVD viral genome of all the
propagated references as well as Iman strains
of BVDV. In an effort to optimize the assay, a
separate RT reaction followed by single PCR
assay was compared to a single tube RT-PCR
assay. Indeed, a separate RT reaction was
found to increase the sensitivity of the BVD
products of approximately 288 bp
amplified with the UTR1 and UTR2
primers. Lane M, the DNA ladder
fragments with sizes of 50, 150, 300,
500, 750 and 1000 bp. Lanes A to E ,
G, H, I and K, the PCR products of
the amplified 288 fragments of the
UTR region of BVDV genome in the
supernatant harvests of infected cells
with Iman strain of BVDV and
collected in time intervals of 8, 12,
20, 24, 30, 48, 72, 96, and 120 hours
post inoculation, respectively. Lane
J, the amplified product from the
supernatant harvest collected at 4
hours PI. Lane F, the amplification
products from the negative control
cells (non infected MDBK cells).
viral RNA detection in both cell-free and cell-
associated harvests of the inoculated cell
culture. BVDV could be detected in the
culture supernatant media shortly after 4 to 8
hours PI which could be the time of eclipse
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