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Elimination of contaminating bovine viral diarrhea virus from bovine respiratory syncytial virus stock
241
Journal of Virological Methods, 6 (1983) 241-244 Elsevier
SHORT
COMMUNICATION
ELIMINATION OF CONTAMINATING BOVINE VIRAL DIARRHEA FROM BOVINE RESPIRATORY SYNCYTIAL VIRUS STOCK* THOMAS
E. TOTH**
and RICHARD
VIRUS
A. HESSE***
Department of Veterinary Science, P. 0. Box 2175, South Dakota State University, Brookings, SD 57007, U.S.A. (Accepted
7 February
During experiments Vero cells supported
1983)
to investigate
the replication
replication
of certain bovine respiratory
of bovine respiratory
virus (BVDV). The selective replication
characteristics
syncytial
viruses, it was observed
that
virus (BRSV) but not bovine viral diarrhea
of these viruses were used to free a BRSV stock from
BVDV contamination.
bovine viral diarrhea
virus
bovine
respiratory
syncytial
virus
elimination
of contaminant
Infection of cattle with bovine viral diarrhea virus (BVDV) is widespread (Potgieter, 1977; Nuttal et al., 1980) although most infections are subclinical (Nuttall et al., 1980). Thus, samples,
which are obtained
either from the respiratory
or the digestive
system of cattle in order to isolate/identify virus(es) other than BVDV, may be contaminated with BVDV. If a specific fluorescent antibody (FA) test for BVDV is not done, the experimenter
may fail to recognize
contamination
with noncytopathic
BVDV, or with a cytopathic strain of BVDV that induces only an abortive infection in cell culture (Fernelius, 1969). Bovine fetal lung cultures and bovine fetal serum may also be contaminated with BVDV (Rossi et al., 1980). If originaly cant mination-free virus strains
are propagated
in the laboratory
with the use of these cultures
and
reagents, they may also become contaminated. These circumstances lead to frequent, inadvertent contamination with BVDV of other bovine viruses or even viruses of other species. In our laboratory we have found that a BRSV strain was contaminated with BVDV (unpubl. data). *
Published Experiment
**
as Journal Station,
Dakota
Address for correspondence: College of Veterinary
***
Series No. South
Ol66-0934/83/0000-0000/$03.00
for publication
State University,
Dr. Thomas
Medicine,
Present address: Salisbury
1857. Approved E. Toth,
Blacksburg,
Laboratories,
Brookings, Research
by the Director,
Center,
Virginia-Maryland
VA 24061, U.S.A.
Inc., Charles
City, IA 50616, U.S.A.
0 1983 Elsevier Science Publishers
Agricultural
SD 57007, U.S.A.
B.V.
Regional
242
Although
BVDV is rapidly
of elimination Elimination problem.
inactivated
by chloroform
can be used only if the contaminated of BVDV from chloroform-sensitive
Cells persistently
infection by continuous and McClurkin, 1981). Our experience
infected passage
with bovine
1963), this method
viruses is a serious
with noncytopathic
syncytial
resistant.
and frustrating
BVDV were not freed of the
in media containing
respiratory
(Dinter,
virus is chloroform
homologous
antibody
(Coria
virus (BRSV) and BVDV indica-
ted that only the former virus replicated in Vero cells. We attempted to use the selective replication characteristics of these viruses to free a BRSV stock from BVDV. A bovine embryonic lung (BEL) cell culture was established in our laboratory by standard methods. The Bel cells were examined by a direct FA test on pasages II 5 and 15 and found to be free of BVDV. Vero cells were obtained from the National Veterinary Services Laboratory (Ames, IA, U.S.A.). indicated by the direct FA test. In the first experiment,
They were free of BVDV as 15, 25 cm2 cell-culture flasks
were seeded with BEL cells (5th passage) and Vero cells (154th passage), respectively, in Eagle’s minimum essential medium (EMEM) containing antibiotics and irradiated fetal bovine serum (FBS). Five flasks of confluent monolayers of both cell-types were inoculated with 0.5 ml/flask of each of the following materials: (a). EMEM containing 2% irradiated FBS to serve as mock-inoculated controls; (b) IO-’ dilution of non-cytopathic BVDV stock (prepared in our BEL cells from New York-l strain); and (c) IO-’ dilution of BRSV stock (prepared in our BEL cells from strain 375, found to be BVDV-contaminated by indirect FA). The inocula were adsorbed for 60 min at 37°C in a humidified, 5% COz-containing atmosphere. The flasks were rinsed three times with EMEM; then EMEM with 2% FBS was added, and the flasks were incubated in the above atmosphere. At 2,3,4,5 and 6 days post-inoculation (PI), the cells from one flask of each cell type-inoculation regimen combination (6 flasks on each day) were trypsinized, seeded on IO-well spot slides, air-dried, fixed in acetone for 10 min at room temperature,
and stained
cence. Results
are shown
uniformly
negative
in Table
by indirect
FA for BRSV- and BVDV-specific
I. Control
cells mock-inoculated
for both viruses. BVDV antigens
fluores-
with EMEM
were not detected
were
by the FA test
in BVDV-inoculated Vero cells on any day PI. However, BVDV antigens were consistently demonstrated from days 3 to 6 PI in both BVDV- and BRSV-inoculated BEL cells. BRSV antigens were recognized by homologous FA test consistently in both BEL and Vero cells that were inoculated with BRSV. The negative FA tests of BVDV-inoculated Vero cells suggested that this cell line was nonpermissive to BVDV replication. However, the possibility remained that BVDV was either latent or of very low titer in the Vero cells, and, therefore, not detected by the FA test. To determine if this was the case, in the second experiment the BVDV-contaminated BRSV stock was passaged once in Vero and then four times in the BEL cells to promote the replication of BVDV in this permissive cell line. Inoculations were done with 0.5 ml of undiluted original BRSV for the first passage and then with culture
243
TABLE
I
Replication
of BVDV and BRSV in Vero and BEL cells FA tests
Days
2
Immunofluorescence*
of
Vero cells inoculated
with
BEL cells inoculated
BVDV
EMEM**
BVDV
BRSV
_
xk
f + + + + + + + + +
BRSV
BVDV BRSV
+
BVDV
3
BRSV 4
BVDV
+
BRSV 5
+
BVDV BRSV
6
BVDV
+
BRSV *
Results of FA tests indicate absence (-), suspicion no attempt
** Virus-free
to quantify
the observation
EMEM with 2% irradiated
(zh), or presence (+)oftypically
by brightness
or proportion
with EMEM
fluorescing cells with
of FA-positive
cells.
FBS.
media for the 4 successive passages in BEL cells. Inoculation and incubation procedures were as described. At each passage, the cells were examined for BVDV- and BRSV-specific fluorescence as in experiment 1. In addition the infectivity of BRSV in culture media of passages 4 and 5 was titrated. Results are shown in Table 2. In the first passage in Vero cells, only BRSV-specific TABLE
2
Effect of back-passaging Passage
no.
in BEL cells on once Vero cell-passaged
Cell type
Harvest
day PI
BVDV-contaminated
Indirect
FA test* for
BVDV
BRSV
BRSV BRSV titer**
I
Vero
4
+
ND***
2
BEL
6
+
ND
3
BEL
4
+
ND
4
BEL
3
+
104.2
5
BEL
3
+
104.7
*
Results of FA tests indicate absence (-) or presence (+) of typically quantify
**
Expressed
***
Not done.
the observation
by brightness
in plaque-forming
or proportion
units per ml.
fluorescing
of FA-positive
cells.
cells with no attempt to
244
fluorescence successive
was seen. While BRSV-specific 4 passages
fluorescence
in BEL cells, BVDV-positive
was also seen in each of the
cells were absent.
The titers of
passages 4 and 5 were typical for this BRSV strain. Results of both experiments indicated
that passaging
in Vero cells eliminated
the BVDV contaminant
BRSV stock, and BVDV was not rescued by passaging
in a susceptible
1 and 2 from the
BEL cell-line.
ACKNOWLEDGEMENTS
Supported in part by USDA, assistance of Gerri McKinney Blacksburg
SEA grant PL-95-113, Section 1433. The secretarial in Brookings and the Word Processing Center in
is acknowledged.
REFERENCES
Coria,
M.F. and A.S. McClurkin,
Dinter,
Z., 1963, Zentrabl.
Fernelius, Nuttall, Potgieter,
A.L., G. Lambert
1981, Am. J. Vet. Res. 42, 647.
Bakteriol. and G.J.
Parasitenkol. Hemness,
P.A., E.J. Stott and L.H. Thomas, L.N.D.,
1977, Bovine Practitioner,
Rossi, C.R., C.R. Bridgman
Infektions
Kr. Hyg.,
Abt.
1 188, 475
1969, Am. J. Vet. Res. 30, 1561.
1980, Res. Vet. Sci. 28, 91. November
75.
and A.K. Kiesel, 1980, Am. J. Vet. Res. 41, 1680.
Journal of Virological Methods, 6 (1983) 241-244 Elsevier
SHORT
COMMUNICATION
ELIMINATION OF CONTAMINATING BOVINE VIRAL DIARRHEA FROM BOVINE RESPIRATORY SYNCYTIAL VIRUS STOCK* THOMAS
E. TOTH**
and RICHARD
VIRUS
A. HESSE***
Department of Veterinary Science, P. 0. Box 2175, South Dakota State University, Brookings, SD 57007, U.S.A. (Accepted
7 February
During experiments Vero cells supported
1983)
to investigate
the replication
replication
of certain bovine respiratory
of bovine respiratory
virus (BVDV). The selective replication
characteristics
syncytial
viruses, it was observed
that
virus (BRSV) but not bovine viral diarrhea
of these viruses were used to free a BRSV stock from
BVDV contamination.
bovine viral diarrhea
virus
bovine
respiratory
syncytial
virus
elimination
of contaminant
Infection of cattle with bovine viral diarrhea virus (BVDV) is widespread (Potgieter, 1977; Nuttal et al., 1980) although most infections are subclinical (Nuttall et al., 1980). Thus, samples,
which are obtained
either from the respiratory
or the digestive
system of cattle in order to isolate/identify virus(es) other than BVDV, may be contaminated with BVDV. If a specific fluorescent antibody (FA) test for BVDV is not done, the experimenter
may fail to recognize
contamination
with noncytopathic
BVDV, or with a cytopathic strain of BVDV that induces only an abortive infection in cell culture (Fernelius, 1969). Bovine fetal lung cultures and bovine fetal serum may also be contaminated with BVDV (Rossi et al., 1980). If originaly cant mination-free virus strains
are propagated
in the laboratory
with the use of these cultures
and
reagents, they may also become contaminated. These circumstances lead to frequent, inadvertent contamination with BVDV of other bovine viruses or even viruses of other species. In our laboratory we have found that a BRSV strain was contaminated with BVDV (unpubl. data). *
Published Experiment
**
as Journal Station,
Dakota
Address for correspondence: College of Veterinary
***
Series No. South
Ol66-0934/83/0000-0000/$03.00
for publication
State University,
Dr. Thomas
Medicine,
Present address: Salisbury
1857. Approved E. Toth,
Blacksburg,
Laboratories,
Brookings, Research
by the Director,
Center,
Virginia-Maryland
VA 24061, U.S.A.
Inc., Charles
City, IA 50616, U.S.A.
0 1983 Elsevier Science Publishers
Agricultural
SD 57007, U.S.A.
B.V.
Regional
242
Although
BVDV is rapidly
of elimination Elimination problem.
inactivated
by chloroform
can be used only if the contaminated of BVDV from chloroform-sensitive
Cells persistently
infection by continuous and McClurkin, 1981). Our experience
infected passage
with bovine
1963), this method
viruses is a serious
with noncytopathic
syncytial
resistant.
and frustrating
BVDV were not freed of the
in media containing
respiratory
(Dinter,
virus is chloroform
homologous
antibody
(Coria
virus (BRSV) and BVDV indica-
ted that only the former virus replicated in Vero cells. We attempted to use the selective replication characteristics of these viruses to free a BRSV stock from BVDV. A bovine embryonic lung (BEL) cell culture was established in our laboratory by standard methods. The Bel cells were examined by a direct FA test on pasages II 5 and 15 and found to be free of BVDV. Vero cells were obtained from the National Veterinary Services Laboratory (Ames, IA, U.S.A.). indicated by the direct FA test. In the first experiment,
They were free of BVDV as 15, 25 cm2 cell-culture flasks
were seeded with BEL cells (5th passage) and Vero cells (154th passage), respectively, in Eagle’s minimum essential medium (EMEM) containing antibiotics and irradiated fetal bovine serum (FBS). Five flasks of confluent monolayers of both cell-types were inoculated with 0.5 ml/flask of each of the following materials: (a). EMEM containing 2% irradiated FBS to serve as mock-inoculated controls; (b) IO-’ dilution of non-cytopathic BVDV stock (prepared in our BEL cells from New York-l strain); and (c) IO-’ dilution of BRSV stock (prepared in our BEL cells from strain 375, found to be BVDV-contaminated by indirect FA). The inocula were adsorbed for 60 min at 37°C in a humidified, 5% COz-containing atmosphere. The flasks were rinsed three times with EMEM; then EMEM with 2% FBS was added, and the flasks were incubated in the above atmosphere. At 2,3,4,5 and 6 days post-inoculation (PI), the cells from one flask of each cell type-inoculation regimen combination (6 flasks on each day) were trypsinized, seeded on IO-well spot slides, air-dried, fixed in acetone for 10 min at room temperature,
and stained
cence. Results
are shown
uniformly
negative
in Table
by indirect
FA for BRSV- and BVDV-specific
I. Control
cells mock-inoculated
for both viruses. BVDV antigens
fluores-
with EMEM
were not detected
were
by the FA test
in BVDV-inoculated Vero cells on any day PI. However, BVDV antigens were consistently demonstrated from days 3 to 6 PI in both BVDV- and BRSV-inoculated BEL cells. BRSV antigens were recognized by homologous FA test consistently in both BEL and Vero cells that were inoculated with BRSV. The negative FA tests of BVDV-inoculated Vero cells suggested that this cell line was nonpermissive to BVDV replication. However, the possibility remained that BVDV was either latent or of very low titer in the Vero cells, and, therefore, not detected by the FA test. To determine if this was the case, in the second experiment the BVDV-contaminated BRSV stock was passaged once in Vero and then four times in the BEL cells to promote the replication of BVDV in this permissive cell line. Inoculations were done with 0.5 ml of undiluted original BRSV for the first passage and then with culture
243
TABLE
I
Replication
of BVDV and BRSV in Vero and BEL cells FA tests
Days
2
Immunofluorescence*
of
Vero cells inoculated
with
BEL cells inoculated
BVDV
EMEM**
BVDV
BRSV
_
xk
f + + + + + + + + +
BRSV
BVDV BRSV
+
BVDV
3
BRSV 4
BVDV
+
BRSV 5
+
BVDV BRSV
6
BVDV
+
BRSV *
Results of FA tests indicate absence (-), suspicion no attempt
** Virus-free
to quantify
the observation
EMEM with 2% irradiated
(zh), or presence (+)oftypically
by brightness
or proportion
with EMEM
fluorescing cells with
of FA-positive
cells.
FBS.
media for the 4 successive passages in BEL cells. Inoculation and incubation procedures were as described. At each passage, the cells were examined for BVDV- and BRSV-specific fluorescence as in experiment 1. In addition the infectivity of BRSV in culture media of passages 4 and 5 was titrated. Results are shown in Table 2. In the first passage in Vero cells, only BRSV-specific TABLE
2
Effect of back-passaging Passage
no.
in BEL cells on once Vero cell-passaged
Cell type
Harvest
day PI
BVDV-contaminated
Indirect
FA test* for
BVDV
BRSV
BRSV BRSV titer**
I
Vero
4
+
ND***
2
BEL
6
+
ND
3
BEL
4
+
ND
4
BEL
3
+
104.2
5
BEL
3
+
104.7
*
Results of FA tests indicate absence (-) or presence (+) of typically quantify
**
Expressed
***
Not done.
the observation
by brightness
in plaque-forming
or proportion
units per ml.
fluorescing
of FA-positive
cells.
cells with no attempt to
244
fluorescence successive
was seen. While BRSV-specific 4 passages
fluorescence
in BEL cells, BVDV-positive
was also seen in each of the
cells were absent.
The titers of
passages 4 and 5 were typical for this BRSV strain. Results of both experiments indicated
that passaging
in Vero cells eliminated
the BVDV contaminant
BRSV stock, and BVDV was not rescued by passaging
in a susceptible
1 and 2 from the
BEL cell-line.
ACKNOWLEDGEMENTS
Supported in part by USDA, assistance of Gerri McKinney Blacksburg
SEA grant PL-95-113, Section 1433. The secretarial in Brookings and the Word Processing Center in
is acknowledged.
REFERENCES
Coria,
M.F. and A.S. McClurkin,
Dinter,
Z., 1963, Zentrabl.
Fernelius, Nuttall, Potgieter,
A.L., G. Lambert
1981, Am. J. Vet. Res. 42, 647.
Bakteriol. and G.J.
Parasitenkol. Hemness,
P.A., E.J. Stott and L.H. Thomas, L.N.D.,
1977, Bovine Practitioner,
Rossi, C.R., C.R. Bridgman
Infektions
Kr. Hyg.,
Abt.
1 188, 475
1969, Am. J. Vet. Res. 30, 1561.
1980, Res. Vet. Sci. 28, 91. November
75.
and A.K. Kiesel, 1980, Am. J. Vet. Res. 41, 1680.