Article

Purified bovine plasma blocking factor decreases Bovine leukemia virus

p24 expression while increasing protein synthesis and transcriptional
activity of peripheral blood mononuclear cells in short-term culture
Marianne J. van den Heuvel, Barbara J. Jefferson, Robert M. Jacobs

Abstract
Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of
infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced
by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly
characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression,
and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The
addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases
in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages
of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.

Résumé
Le virus de la leucémie bovine (BLV) induit une infection persistante des cellules B entraînant une expansion polyclonale des cellules B chez
le tiers des bovins affectés et des lymphosarcomes dans moins de 5 % des bovins affectés. Bien que le BLV soit difficile à détecter in vivo, il
est facilement produit par des lymphocytes en culture et cette production est diminuée lors de l’ajout de plasma bovin. Ce phénomène est
attribué à une substance pauvrement caractérisée, le facteur plasmatique bloquant (PBF). Nous avons évalué les effets du plasma bovin sur
la viabilité cellulaire et l’expression de la p24 du BLV, et les effets du PBF purifié sur la synthèse protéique et l’expression génique de
lymphocytes bovins lors de culture de courte durée. L’ajout de 25 % de plasma ou de PBF semi-purifié aux cultures n’avait aucun effet
significatif sur la viabilité cellulaire mais entraînait une réduction significative de la production de p24 du BLV et une augmentation
significative de la synthèse protéique de novo. À l’aide d’une puce à ADN humaine, il a été déterminé que l’expression de l’ARN messager
de 83 gènes impliqués dans la division cellulaire, le métabolisme cellulaire et la régulation génique était augmentée.
(Traduit par Docteur Serge Messier)

Introduction
Bovine leukemia virus (BLV) induces a persistent infection primar-
ily in B-cells (1). The BLV genome encodes for a trans-regulatory
protein, Tax, that efficiently induces viral transcription and transac-
tivates cellular genes, which, in the case of BLV-infected cattle, may
lead to dysregulation of B-cells (2). An expression of this dysregula-
tion may be persistent lymphocytosis (PL), a non-neoplastic expan-
sion of B-cells, which is observed in approximately one-third of
naturally or experimentally infected cattle.
Following a prolonged incubation, less than 5% of BLV-infected
adult cattle develop neoplasia of CD5 B-cells termed lymphosar-
coma (3). Persistent lymphocytosis (PL) may be considered a pre-
neoplastic change since it precedes lymphosarcoma in approximately
two-thirds of affected cattle.
Despite being replication-competent, BLV is quiescent in vivo (4).
Characteristics of Tax and other viral regulatory elements are impor-
tant in repressing viral expression and contributing to latency (5);
however, the latent state is rapidly reversed when infected lympho-
cytes are cultured in vitro (6). Host cell factors, such as interleukin
(IL)-6 and tumor necrosis factor (TNF)-, have also been shown to
potently suppress BLV production in vitro (7).
A marked decrease in BLV production in vitro has been demon-
strated to be induced by a poorly defined ‘plasma blocking factor’
(PBF), which inhibits transcription of the integrated provirus (8–10).
Similar activity has been described in the plasma of BLV-infected
sheep (11) and patients infected with Human T-cell leukemia virus-1
(12) or Human immunodeficiency virus (HIV)-1 (13).
The PBF is constitutively expressed since it is found in the plasma
of all cattle regardless of BLV status (14,15). The phenomenon is
different in sheep since only BLV-infected individuals display PBF
activity (8). The addition of bovine plasma to short-term cultures of
peripheral blood mononuclear cells (PBMCs), in either autologous
or homologous culture conditions, is sufficiently potent as to over-
come the up-regulatory effects of fetal bovine serum (FBS) or lectins
on BLV p24 production (15). Cross-species activity is not evident
since bovine plasma has no effect on the production of murine or
feline leukemia viruses growing in monolayer cell cultures. The PBF
activity is either not present in serum or its activity is less in serum
compared with plasma (10,15). Platelet lysates inhibit PBF activity,

Department of Pathobiology, University of Guelph, Ontario N1G 2W1.

Dr. van den Heuvel’s current address is Department of Pediatrics, University of Western Ontario, London, Ontario N6C 2V5.
Address all correspondence and reprint requests to Dr. Marianne van den Heuvel; telephone: (519) 685-8500 ext 55070; fax: (519) 685-8186;
e-mail: mvandenh@uwo.ca

186 The Canadian Journal of Veterinary Research 2005;69:186–192

likely accounting for decreased activity in serum (16). Absence of
activity in the Vesicular stomatitis virus plaque reduction assay,
similar PBF activity in BLV-infected and non-infected cattle and
decreased activity with exposure of plasma to platelet lysate indicate
that PBF is not interferon (IFN)- or an immunoglobulin molecule.
A molecular size of 150 kDa has been suggested (9,10).
Seroepidemiological studies have shown that retroviral infections
are common in cattle yet overt disease is uncommon (1). In contrast,
disease is more prevalent in those species where retroviral infections
are productive. Undoubtedly, the profound latency observed with
BLV is a key factor in limiting disease expression. The PBF has the
potential to, at least partially, explain BLV latency, but its constitutive
expression suggests a larger homeostatic role.
There is relatively little known about the cell biology of PBF. In
this preliminary study, we sought to investigate the effects of bovine
plasma on cell viability, purified PBF on protein synthesis, and gene
expression in short-term cultures of PBMCs.
Materials and methods
Animals
Bovine leukemia virus serostatus was assessed by repeated agar gel
immunodiffusion tests. Blood samples from 8 naturally infected
BLV-seropositive (BLV) Holstein cows; 4 with a normal lymphocyte
count (asymptomatic), 4 with PL (lymphocytes in excess of 8000/L
for  1 y), and 3 BLV-seronegative (BLV) cows were obtained from
a commercial dairy herd.
Peripheral blood mononuclear cell culture
Anticoagulated blood was centrifuged for 25 min at 800  g and
20°C. The buffy coat was aspirated and diluted 1:5 with phosphate
buffered saline solution (PBSS, 0.01 M, pH 7). Fifteen milliliters of
buffy coat cell solution were layered over 15 mL of Ficoll (Histopaque
1.077; Sigma Aldrich, Mississauga, Ontario) and centrifuged at
400  g for 30 min at 20°C. The PBMCs were removed and washed
3 times with PBSS. The cells were diluted to a concentration of
2  106 viable cells per mL of RPMI supplemented with 10% heat
inactivated FBS; L-glutamine (0.3 g/L); and 0.02% amikacin, with
or without 25% heat inactivated autologous plasma (v/v). Cultured
cells (2 mL) were incubated at 37°C and 5% CO2 for 24 h. Cell cul-
tures were done in triplicate.
Cell viability, when cultures were initiated and at 24 h, was
assessed by trypan blue exclusion and manual counting. These stud-
ies were replicated 8 times over the duration of the study.
Bovine leukemia virus p24 production
After a 24 h culture period, cells were collected by centrifugation,
washed, and lysed with a 10% aqueous solution of n-octyl- -D-
glucopyranoside (Sigma Aldrich). The lysate was diluted 1:10 in
sterile PBSS. Clarified cellular lysate was applied in triplicate to wells
of a 96-well plate (Nunc Immunosorb; Life Technologies, Mississauga,
Ontario), which had been coated with a monoclonal anti-BLV p24
(4’G9, kindly provided by Dr. D. Portetelle) for 6 h, based on an
established protocol (15,17,18). Doubling dilutions of affinity-purified
BLV p24 were used to construct a standard curve. Following an
2000;64:0–00
overnight incubation at 4°C, 100 L of an antibody solution, contain-
ing 200  g each of 2 (2’C1 and 4’F5) monoclonal peroxidase-
conjugated antibodies to BLV p24 (generously provided by
D. Portetelle), were added to each well. The plate was incubated for
1 h and washed before initiating the color reaction by the addition
of tetramethylbenzidine. The color development was measured
spectrophotometrically at 450 nm.
Purification of PBF
Briefly, whole plasma was heated to 65°C for 10 min to precipitate
fibrin, then centrifuged at 2500  g for 10 min at 4°C. The superna-
tant was decanted and 20% saturated ammonium sulphate was
added to the plasma at 4°C, while stirring continuously for 10 min.
The solution was then centrifuged at 5000  g and 4°C for 30 min.
The supernatant was collected and subjected to further precipitation
with 30% saturated ammonium sulphate, stirred, and centrifuged
as before. The supernatant was discarded and the pellet resuspended
in sterile water. The plasma fraction was dialyzed for 24 h against
3 changes of buffer (150 mM KCl, 50 mM KH 2PO4, pH 7). After
equilibration, this plasma fraction was applied to an fast protein
liquid chromatography (FPLC) column containing Sepharose Blue
(Pharmacia, Mississauga, Ontario) equilibrated into the same buffer.
The protein was eluted with a step-wise increase in the KCl concen-
tration. The fraction, which eluted from the column with the addition
of 500 mM KCl, was collected, de-salted, equilibrated into a buffer
of 100 mM KCl, and concentrated to 10 mg/mL. This was applied
to a Superose 6 column (Pharmacia) and eluted with the same buffer.
‘Purified PBF’ was eluted from the column during the first 25 min
and concentrated to 10 mg/mL.
Protein synthesis
Protein synthesis was measured by quantifying the uptake of
3H-leucine
in short-term cultures as described previously (19). The
PBMCs were isolated from BLV, BLV asymptomatic, and BLV
with PL cows and were resuspended at a concentration of 5  106
viable cells per mL of leucine-free RPMI (Sigma Aldrich) supple-
mented with L-glutamine (0.3 g/L), L-lysine (0.04 g/L), L-methionine
(0.015 g/L), and Na(CO3)2 (2 g/L). Eight replicate lymphocyte assays
were established in round-bottomed 96 well plates to which 25 L
of cell suspension and 25 L of culture medium alone or culture
medium with 250 g of either bovine serum albumin (BSA) or puri-
fied PBF was added. After incubation at 37°C in a 5% CO 2 atmo-
sphere for 1, 4, 8, 16, 20, or 24 h, the plates were pulsed with 0.4 Ci
of 3H-L-leucine (Sigma Aldrich) in 10 L of RPMI, then cultured for
1 h. The plate was then frozen to stop additional incorporation of
3H-leucine. The plate contents were aspirated onto glass fibre filters
and washed to remove unincorporated 3H-leucine. The filters were
dried for 1 h at 60°C then counted. The mean counts per minute
(cpm) were calculated for each of the treatments for each sero-status
group of cows. The experiment was done in triplicate.
DNA microarray analysis
The PBMCs from 3 cows; 2 BLV with PL and 1 uninfected, were
cultured for 16 h in RPMI with or without the addition of purified
PBF. At the termination of the culture, cells were pelleted, washed
in PBSS, pH 7, 3 times, then RNA was extracted using a commercial
The Canadian Journal of Veterinary Research 187
 Table I. Effect of 25% (v/v) autologous plasma on cell number, viability and Bovine
leukemia virus (BLV) p24 production in short-term cultures (mean ± s, n = 8) of peripheral
blood mononuclear cells

Time in Total number of Cell viability at

culture Plasma BLV non-adherent cells termination BLV p24
(hours) added status ( 105) (%) (ng /105 viable cells)
0  Neg 33.8 ± 3.5 98.7 ± 0.4 Not detectable
0  AS 35.6 ± 2.5 98.9 ± 0.5 Not detectable
0  PL 35.1 ± 1.1 98.8 ± 0.5 Not detectable
24  Neg 37.2 ± 12.7 71.3 ± 8.7 Not detectable
24  AS 33.4 ± 10.5 69.7 ± 16.2 7.3 ± 3.1
24  PL 31.8 ± 13.0 60.8 ± 19.5 36.8 ± 20.1
24  Neg 41.2 ± 11.2 69.2 ± 12.3 Not detectable
24  AS 37.8 ± 14.5 71.9 ± 15.7 Not detectable
24  PL 39.5 ± 14.6 58.6 ± 21.3 6.5 ± 3.5a
a Significantly different than medium alone at 24 h of culture (P  0.01)

Neg — negative; AS — asymptomatic; PL — persistent lymphocytosis

kit (RNAeasy; Qiagen, Mississauga, Ontario). Samples of the isolated
RNA were diluted in water and quantified by absorbence measure-
ments at 260/280 nm. Quality of RNA was assessed using a
BIO analyzer (Agilent, Mississauga, Ontario). Five micrograms of
RNA from each sample were reverse transcribed in the presence of
amino allyl dUTP (Sigma Chemical Company, Oakville, Ontario).
Alexa fluor 555 and Alexa fluor 647 (Molecular Probes, Eugene,
Oregon, USA) were coupled to amino allyl dUTP in cDNA from
PBF-treated and untreated cells, respectively. Fluorescently labeled
samples were then hybridized to slides containing a 19k human
array (Single Spot H19K, version 8; Ontario Cancer Institute, Toronto,
Ontario). The hybridized microarrays were then scanned (Genepix
4000 A Scanner; Axon, Foster City, California, USA).
Statistical analysis
Data were summarized using descriptive statistics. Treatment
effects were assessed using the one way analysis of variance
(ANOVA). Differences were considered significant if P  0.05.
Microarray spots were quantified (Genepix Pro 3.0; Axon), normal-
ized using the lowest algorithm (GeneTraffic; Iobion Informatics,
LaJolla, California, USA), and analyzed statistically (Significance
Analysis of Microarray; SAM, Stanford University, Stanford,
California, USA) in a one-class response format.
able upon the addition of plasma to the cultures. Bovine leukemia
virus p24 was not detected in the BLV cows.
Purification of PBF
Purified PBF, having a molecular weight of approximately
155 kDa and a pI of 5.5 (20), was produced by a series of physico-
chemical and chromatographic steps and used in the protein synthe-
sis and DNA microarray studies. The biological activity of purified
PBF was maintained since it decreased BLV p24 expression in
cultures of PBMCs by approximately 80% of the activity of whole
plasma (data not shown).
Protein synthesis
The PBMCs isolated from 3 Holstein cows in each group having
a sero-status of BLV, BLV asymptomatic, and BLV with PL, were
cultured in leucine-free medium, then pulsed with 3H-leucine.
Lymphocytes cultured in medium or in medium with added BSA
(5 mg/mL), and regardless of BLV status, showed only a small
amount of protein synthesis over 16 h in culture (Figure 1), which
was sustained at 24 h (data not shown). However, in cultures of
PBMCs with added purified PBF (5 mg/mL) a marked increase in
protein synthesis occurred as early as 12 h post inoculation, regard-
less of serostatus. Protein synthesis in response to PBF peaked at
16 h, reaching an increase of 9-fold over media alone for the cells

Results
Cell viability and BLV p24 production
At the initiation of short-term cultures, PBMCs from all cows had
a viability of approximately 98%. There were no significant differ-
ences in cell counts and viability (67 ± 5.3%) between culture with
or without added plasma (Table I). The addition of plasma to the
cultures resulted in a significant decrease in the amount of BLV p24
produced (P  0.01). Cell viability and cell counts in cultures from
the BLV and BLV asymptomatic cows were not significantly dif-
ferent from those observed in the BLV cows with PL (Table I). Bovine
leukemia virus p24 in the BLV asymptomatic cows was approxi-
mately 20% of that produced in the cow with PL and was undetect-
from the BLV cows, 18-fold for the BLV asymptomatic cows, and
13-fold in the cells from the BLV cows with PL. The incorporation
of 3H-leucine in cultures supplemented with purified PBF was sig-
nificantly greater than the medium control and the BSA control for
all cows after 12 h (P  0.01).
DNA microarray analysis
The human array used in this experiment was comprised of
19 000 genes. The bovine cRNA was able to recognize and bind to
13 491 out of 14 793 genes on the array (91% bound). The array
results were pre-scanned to detect and flag unusual hybridization
spots. A delta value of 0.60584 was set to minimize the false discovery
rate. The upper cut-off was 1.59241 and the lower cut-off was infin-
ity. Using computer software (SAM) to scan the arrays, the genes

188 The Canadian Journal of Veterinary Research 2000;64:0–00


Figure 1. The effects of culture conditions and Bovine leukemia virus (BLV)
serostatus on protein synthesis as determined by the uptake of 3H-leucine.
A, peripheral blood mononuclear cells (PBMCs) isolated from BLV-serone-
gative cows (n = 3); B, asymptomatic BLV-seropositive cows (n = 3); and
C, BLV-seropositive cows with persistent lymphocytosis (n = 3). The PBMCs
were cultured in leucine-free medium (), leucine-free medium with bovine
serum albumin (BSA) (5 mg/mL) () or leucine-free medium with added
purified plasma blocking factor (PBF) (5 mg/mL) (). Data points represent
the mean (± sx̄) in which each determination was the result of 8 replicate
cultures. Commencing at 12 h, cultures supplemented with purified PBF
were significantly greater (P  0.05) than control cultures.
selected were the most highly up or down regulated in PBMCs
grown in medium supplemented with purified PBF relative to those
grown in medium alone, but which are also consistent between the
3 biological replicates. One hundred and nine genes were found to
be significantly up-regulated, of which 26 were flagged in at least
1 sample as irregular binding and were thus discarded. Table II lists
the remaining 83 significantly upregulated genes, grouped according
to function. The table lists the gene name, the delta score for that
gene, and the gene product. The delta score, which is calculated in
a one way analysis, is related to the significance of that gene’s expres-
sion. Thus, as the delta score increases, so does the significance (21).
2000;64:0–00
The largest group, comprising 16 genes, were the genes related to
catabolism, or having enzymatic functions. Next were genes related
to transcription or DNA repair (9 genes), followed by genes involved
in immune response (6 genes), intracellular signalling (6 genes), and
ion transport (5 genes). Interestingly, while each of the individuals
had a few down-regulated genes, these differed between individuals,
thus no significantly downregulated genes common to the group
were found.
Discussion
In this preliminary study, the effects of bovine plasma on cell viabil-
ity, protein synthesis, and mRNA expression were investigated. As
anticipated, the addition of 25% plasma significantly down-regulated
BLV p24 expression, which was not associated with significant
changes in cell count or viability (10,11,17). The PBF inhibits BLV
expression via transcriptional control (9) and, as the present study
confirms, it does so independent of gross changes in cell kinetics.
The aim of the 3H-leucine experiment was to determine whether
the down-regulation of BLV p24 in cultures supplemented with
plasma resulted from a global depression of protein synthesis.
Surprisingly, the addition of purified PBF to cultures potently
stimulated protein synthesis despite inhibition of BLV p24 expres-
sion. Furthermore, the effect was also present in the PBMC from the
BLV cows. Since PBF is present in all cattle and the data here sug-
gest it increases protein synthesis regardless of BLV status, then PBF
may have importance in homeostasis as well as in host susceptibil-
ity and BLV latency.
Approximately 70% of cattle that become BLV-infected are
‘nonprogressors’ in that they have a quiescent infection and
remain asymptomatic for life. A minority of BLV-infected cattle
are ‘progressors’ and develop PL. About two-thirds of cattle with
lymphosarcoma have a history of PL. Although multiple host
factors are likely associated with the development of PL, it is
clear the cytokine milieu is important. In the paradigm of cattle
progressing from being BLV asymptomatic, to BLV with PL,
and finally BLV with lymphosarcoma, there is a shift away from
initial prevalence of type 1 cytokines (IL-2, IL-12, IFN- ) to IL-10
later on (22,23). Furthermore, HTLV and BLV genomes include
regulatory proteins, which not only stimulate viral transcription and
co-ordinate virus packaging, but also act as regulatory elements of
cellular genes, particularly those that act as stimulators or repres-
sors of the immune system, such as IL-2 (22), the IL-2 receptor
(24,25), transforming growth factor- (26), TNF-, and the  light
chain (27).
Since it was evident that PBF stimulated protein synthesis and
others have demonstrated an association of cytokine alterations on
BLV p24 production (7,22,23), we felt that microarray analysis of the
effects of PBF on bovine PBMCs would potentially be an efficient
tool to probe changes in transcription. A bovine microarray was
unavailable, consequently a human 19K DNA microarray was uti-
lized. We reasoned that poor homology would result in the decreased
intensity or absence of a signal, however, under the conditions of
the experiment, relative changes in signal intensity associated with
the addition of plasma were sought. However, the bovine cRNA
demonstrated good ability to bind to the human probes.
The Canadian Journal of Veterinary Research 189
Table II. Genes up-regulated in peripheral blood mononuclear cells from 3 Bovine leukemia virus (BLV)-seropositive cows cultured
with purified plasma blocking factor (PBF), as compared with those cultured in medium
Gene Ratio Product Gene Ratio Product
Extracellular matrix interactions CBFA2T1 1.833562 core-binding factor, runt domain, alpha
LOX 3.835313 lysyl oxidase subunit 2; cyclin D-related
Protein transport/binding NAB1 1.785353 NGFI-A binding protein 1
MMAA 2.864399 methylmalonic aciduria (EGR1 binding protein 1)
(cobalamin deficiency) type A SALL2 1.685080 sal-like 2 (Drosophila)
WAS 2.552505 Wiskott-Aldrich syndrome CSNK2A2 1.659878 casein kinase 2, alpha prime polypeptide
(eczema-thrombocytopenia) CHD1L 1.637005 chromodomain helicase DNA binding
LMO7 1.990107 LIM domain only 7 protein 1-like
CDH5 1.86503 cadherin 5, type 2, VE-cadherin RPL31 1.605028 ribosomal protein L31
(vascular pithelium) Immune response
FCN1 1.847599 ficolin (collagen/fibrinogen domain CCL3 2.018784 chemokine (C-C motif) ligand 3
containing) 1 HLA-DQB1 1.766330 major histocompatibility complex, class II,
VPS4B 1.613197 vacuolar protein sorting 4B (yeast) DQ beta 1
Enzyme/catabolism CR1L 1.702018 complement component (3b/4b) receptor
LIPC 2.653559 lipase, hepatic 1-like
CES2 2.383959 carboxylesterase 2 (intestine, liver) HLA-DPB1 1.667414 major histocompatibility complex, class II,
OSBPL10 2.292379 oxysterol binding protein-like 10 DP beta 1
CSNK1G1 2.196898 casein kinase 1, gamma 1 SNN 1.621415 stannin
CCNK 2.164055 cyclin K C5 1.615184 complement component 5
OAZ2 2.137920 ornithine decarboxylase antizyme 2 Cell cycle
MOCS3 2.076013 molybdenum cofactor synthesis 3 SSR1 1.782304 signal sequence receptor, 
GM2A 2.018321 GM2 ganglioside activator (translocon-associated protein )
ALDH3B1 1.891641 aldehyde dehydrogenase 3 family, CUL4B 1.728568 cullin 4B
member B1 CD81 1.595990 CD81 antigen (target of antiproliferative
IMPA1 1.765316 inositol(myo)-1(or 4)-monophosphatase 1 antibody 1)
UROS 1.72044 uroporphyrinogen III synthase CUL1 1.592410 cullin 1
(congenital erythropoietic porphyria) Cell mobility
GPD1 1.707899 glycerol-3-phosphate dehydrogenase 1 MYO5 1.650929 myosin VA (heavy polypeptide 12, myoxin)
(soluble) UTRN 1.623126 utrophin (homologous to dystrophin)
NT5C2 1.671468 5’-nucleotidase, cytosolic II
USP47 1.642920 ubiquitin specific protease 47 Hypothetical proteins/Unknown function
CAS1 1.635062 O-acetyltransferase C9orf37 2.711232 chromosome 9 open reading frame 37
PSMF1 1.633274 proteasome (prosome, macropain) inhibitor NA 2.490859 Transcribed sequence with weak similarity
subunit 1 (PI31) to protein sp:P39188 (H. sapiens)
ALU1_HUMAN Alu subfamily J
Cell membrane/Tight junctions C20orf104 2.473668 chromosome 20 open reading frame 104
CLDN4 2.578488 claudin 4 C22orf4 2.119500 chromosome 22 open reading frame 4
ARL6IP 2.003572 ADP-ribosylation factor-like 6 interacting SRRM2 2.036568 serine/arginine repetitive matrix 2
protein MGC955 1.994509 hypothetical protein MGC955
Intracellular signalling NA 1.932072 Transcribed sequence with moderate
HSPC163 2.563149 HSPC163 protein similarity to protein ref:NP_060265.1
CD48 2.437316 CD48 antigen (B-cell membrane protein) (H. sapiens) hypothetical protein FLJ20378
CGI-141 2.162565 CGI-141 protein KIAA0543 1.902394 KIAA0543 protein
TP53AP1 2.122240 TP53 activated protein 1 C6orf133 1.900559 chromosome 6 open reading frame 133
NRG1 1.632979 neuregulin 1 NA 1.891882 CDNA FLJ41736 fis, clone HLUNG2019058
TRIP12 1.601443 thyroid hormone receptor interactor 12 C21orf66 1.883091 chromosome 21 open reading frame 66
Ion transport KIAA0773 1.865317 KIAA0773 gene product
FRDA 2.538091 Friedreich ataxia NA 1.857846 MRNA full length insert cDNA clone
RER1 2.230386 RER1 homolog (S. cerevisiae) EUROIMAGE 1635266
PLP2 2.037553 proteolipid protein 2 FLJ23467 1.847888 hypothetical protein FLJ23467
(colonic epithelium-enriched) MYH14 1.832412 myosin, heavy polypeptide 14
SLC17A2 1.820366 solute carrier family 17 (sodium PRO2730 1.782297 hypothetical protein PRO2730
phosphate), member 2 FLJ12892 1.755869 hypothetical protein FLJ12892
MT1B 1.782754 metallothionein 1B (functional) MGC35366 1.752168 hypothetical protein MGC35366
KIAA0773 1.703299 KIAA0773 gene product
Transcription/DNA repair FLJ10204 1.694151 hypothetical protein FLJ10204
RAD51L1 2.030414 RAD51-like 1 (S. cerevisiae) FLJ36175 1.688923 hypothetical protein FLJ36175
ZNF297B 1.988741 zinc finger protein 297B NA 1.663994 CDNA FLJ44242 fis, clone
UBE2V1 1.877336 ubiquitin-conjugating enzyme E2 variant 1 THYMU3008672

190 The Canadian Journal of Veterinary Research 2000;64:0–00

 Several genes among those most highly amplified by the addition
of PBF, have important functions in cell division, signal transduction,
and regulation of gene expression. The gene for lysyl oxidase (LOX)
was the most up-regulated gene. Lysyl oxidase is the main collagen
and elastin cross-linking enzyme in involved in fibrosis by catalyz-
ing the formation of the lysine-derived aldehyde (28). Also of inter-
est was ficolin1, a leukocyte-derived protein which also binds
elastin (29). Elastin confers elasticity to many tissues, including the
vascular system, thus up-regulation of these genes indicates a pos-
sible role for PBF in maintaining blood vessel structure.
Only 6 immune-response genes were identified as up-regulated
by PBF. Two of these were major histocompatibility complex (MHC)
class II genes involved in antigen presentation (DQ 1 and DP 1),
suggesting that PBF may enhance the ability of antigen presenting
cells in peripheral blood to activate immune response. C-C chemo-
kine 3, a chemokine associated with in vitro inhibition of HIV infec-
tion, but not associated with progression or inhibition of infection
in vivo (30), was up-regulated by PBF. Other immune response
proteins up-regulated include complement component 5 and the
receptor for C3b/4b.
The largest group of up-regulated genes included genes encoding
enzymes involved in catabolism. In HIV-infected patients receiving
anti-viral therapy, an increase in lipid and cholesterol due to reduced
activity of lipase is not uncommon (31). Other groups include genes
involved in intra-cellular signalling, ion transport, protein binding,
and DNA repair. Thus, from this preliminary investigation, it appears
that PBF has a multiplicity of effects on transcription and here we
have identified several groups of genes worthy of further study.
In summary, these preliminary data provide evidence that a
factor(s) in bovine plasma down-regulated expression of BLV by an
unknown mechanism, but nevertheless strongly up-regulated protein
synthesis. Adding plasma to PBMC cultures did not significantly
alter cell viability suggesting that an increased rate of cell death did
not account for the decreased BLV p24 production. Microarray
analysis showed numerous alterations in transcription, which sug-
gests there are a complexity of factors induced by PBF and this
should serve as a new focus for studying BLV-related latency and
pathogenesis. There are a series of interesting candidate genes that
may play pivotal roles in mediating the phenomenon of PBF.
Additional studies with larger numbers of cattle will be needed to
assess genes for their primary or secondary importance.
Acknowledgments
This research was supported by grants from the Natural Sciences
and Engineering Research Council of Canada and the Ontario
Ministry of Agriculture, Food and Rural Affairs. Marianne van den
Heuvel was the recipient of a Dairy Farmers of Ontario Doctoral
Scholarship. The authors thank Dr. D. Portetelle for his willingness
to share unique reagents. The work of Doug Dean in preparing the
microarray analysis is gratefully acknowledged.
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