Embed Size (px)
Citation preview
Detection and molecular characterization of bovine leukemia virus in various
regions of Iran
Monireh Kazemimanesha, Omid Madadgarb,c*, Falko Steinbachd, Bhudipa Choudhurye,
Kayhan Azadmanesha*.
a Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran
b Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.
c Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing,
Michigan, USA.
d Virology Department, Animal and Plant Health Agency, Addlestone, United Kingdom.
e OIE Reference Laboratory for EBL, Department of Virology, Animal and Plant Health Agency,
Weybridge, UK.
*Address correspondence to Omid Madadgar, [email protected] , or Kayhan Azadmanesh,
Keywords: Bovine Leukemia Virus, Molecular Epidemiology, Phylogeny, Iranian Isolate.
Repositories: 24 sequences of Iranian Isolates were submitted to GenBank (accession no.
MG204538- MG204561)
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Abstract
Purpose: Bovine leukemia virus (BLV) infects cattle worldwide, imposing an economic impact
on the dairy cattle industry. The purpose of this study was to evaluate the molecular
epidemiology of BLV in Iran.
Methodology: Blood samples taken from 280 cows aged over 2 years old from 13 provinces of
Iran were used for leukocyte count and blocking ELISA. Genomic DNA was extracted from the
peripheral blood leukocytes of BLV-infected samples and FLK cells to perform PCR of partial
env, rex and tax genes and LTR region. The PCR products were sequenced, the phylogenetic tree
of each gene was constructed, and nucleotide and amino acid sequence pair distances were
calculated.
Results: The frequency of BLV was 32.8% among animals and was 80% among provinces. In
BLV seropositive animals, the rate of persistent lymphocytosis (PL) was 36.9%. The constructed
phylogenetic trees showed the presence of two BLV genotypes (1 and 4) in Iranian strains. In
accordance with the results of previous studies, our results showed that the env gene was more
variable than previously thought, the Rex protein could withstand more amino acid changes
compared to the Tax protein, and no significant differences were observed in average changes of
the nucleotide of these genes between clinical stages.
Conclusions: Comparison of our data with previous studies on seroprevalence of BLV, indicates
an increase in the frequency of this infection in Iran. This is the first study report of the presence
of BLV genotype 4 in Iranian farms. These findings may have an important role in the control
and prevention of BLV infection in Iran and other countries.
2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Introduction
Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic member of the Retroviridae family
belonging to the genus Deltaretrovirus which also includes human T lymphotropic virus 1, 2, 3
and 4 (HTLV-1, -2, -3 and -4) and simian T lymphotropic virus1, 2, 3 and
5 (STLV-1, -2, 3 and -5). BLV infects cattle worldwide and is the causative agent of enzootic
bovine leukosis (EBL) which is an important agricultural problem (1).
BLV is a lifelong infection characterized by a long period of viral latency and absence of
viremia. Most BLV infections are asymptomatic in an aleukemic stage (AL) and are recognized
only by serological testing. Among infected cattle, about 30% develop persistent lymphocytosis
(PL), characterized by a benign polyclonal proliferation of B-cells. Less than 5% of the infected
animals develop lymphosarcoma (2). Despite the low incidence of diseases associated with BLV,
the infection does cause important economic losses in the livestock industry (3) such as the costs
of control and eradication programs, culling of cattle with lymphosarcoma, decreased lifespan,
loss of production potential, and restrictions on the export of cattle, semen, and embryos to
countries that maintain BLV control programs. Moreover, BLV infection may impair the
immune system leading to opportunistic infections (1, 2). On the other hand, no effective vaccine
or therapy has been developed yet.
BLV can infect various immune cell populations, including CD5+ IgM+ and CD5− IgM+ B-
cells; CD2+, CD3+, CD4+, CD8+, and γ/δ T-cells; monocytes; and granulocytes in the
peripheral blood and also lymphoid tissues and mammary gland cells of cattle. During infection,
the provirus integrates into the genome and therefore BLV can be found in the cellular fraction
of various body fluids (4). Disease transmission between cattle occurs via exposure to infected
lymphocytes in the blood from parturition, contaminated surgical instruments, contaminated
3
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
needles for injection, rectal palpation and bloodsucking insects. Prenatal and milk (colostrum or
mature milk) transmission have been demonstrated in BLV infection (2, 3, 5, 6).
All cattle breeds are susceptible to BLV infection. The prevalence is higher in large herds than in
smaller herds and there is no relationship between sex and BLV infection (2, 7).
As a retrovirus, the genome of BLV consists of two identical linear positive-sense single-
stranded RNA molecules that are transcribed to a double-stranded DNA by the reverse
transcriptase enzyme, and then integrated into the B-cell genome as a provirus. BLV has two
identical long terminal repeats (LTRs), which possess a U3 region, an unusually long R region
and a U5 region. Transcription of BLV genes initiates at the U3/R junction in the 5'LTR and is
regulated by cellular transcription factors for which several binding sites have been identified in
the LTR, by the viral trans-activator TAX and by the chromatin status of the BLV provirus(8).
The presence of BLV antisense transcription implies that the BLV LTR is capable of driving
transcription in both directions(9). Ten mature miRNAs from five different pre-microRNA genes
are detected in BLV infected B-cells. Recent studies suggest that the miRNAs are essential
players in viral persistence and oncogenesis(10). BLV expresses antisense transcripts, AS1 and
AS2, driven by 3'LTR-dependent TATA-less promoter activity and constitutively produced in
leukemic cells. AS1 and AS2 (like HBZ of HTLV-1) play an important role in the life cycle and
oncogenic potential of the virus. In addition, BLV strongly expresses RNA polymerase III-
dependent microRNAs that overlap AS1 and contribute to about 40% of microRNAs in the
tumor cell(11, 12).
The BLV genome contains the gag, pol and env structural genes as well as, regulatory genes
including the tax, rex, R3 and G4. The env gene encodes a polyprotein precursor (gp72), which is
cleaved to form the gp51 surface (SU) and gp30 transmembrane (TM) glycoproteins. The Env
glycoprotein is essential to viral infectivity and syncytium formation and is also a target for
4
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
neutralizing antibodies(13, 14). The tax gene encodes a 34 kDa protein (p34) that functions as a
transactivator of the LTR promoter region(15). In addition to its role as a transcriptional
activator, Tax can cooperate with the Ha-ras oncogene to induce malignant transformation of rat
embryo fibroblasts(16). The rex gene, which has 420 bp in common with the tax gene, encodes
Rex, a nuclear phosphoprotein that acts post-transcriptionally through cis-acting elements in the
LTR and binding to and stabilizing viral RNAs, that regulate the expression levels of genes
encoding virion components(17). It also has a nuclear export signal and acts to facilitate the
export of intron-containing viral RNAs(18).
Analyses of the BLV env gene in strains isolates obtained from different geographical areas have
demonstrated significant sequence conservation(19, 20). Nevertheless, at least 10 genotypes have
been identified based on the env gene (21, 22).
BLV infects dairy and beef cattle in Iran and the prevalence of the infection has risen(23).
However, few studies have investigated the genetic variability of BLV in Iran. An previous study
carried out in 2007 using partial BLV env sequences of strains BLV isolates from five provinces
of Iran, revealed that Iranian isolates belonged to genotype 1(24). To improve our knowledge of
the state of BLV in Iran, we collected Iranian BLV strains isolates from 13 provinces and
characterized them by phylogenetic analysis of partial sequences of the structural env gene, as
well as regulatory rex and tax genes and LTR region.
Materials and Methods
Bovine samples
5
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
This work was approved by the Ethical Committee of the Faculty of Veterinary Medicine of
University of Tehran. In this project, blood samples were collected from farms and
slaughterhouses. In farms, blood samples were taken at the time of periodic blood sampling for
animal health checkup by farms’ vets. In slaughterhouses, blood samples were taken after using
electric narcosis.
A total of 280 blood samples were collected from cows aged over 2 years from 10 provinces of
Iran in different regions and environments during August 2010 to January 2012. In addition, 9
BLV positive samples from 3 provinces of Iran were kindly provided by Mabna Lab (Table 1
and Figure 1).
Peripheral blood was aseptically obtained from jugular vein with and without EDTA. Samples
were transported to the laboratory at 4°C. For serum collection, blood without EDTA was kept
cool to allow clotting and tubes were centrifuged at 1500 g for 15 minutes. Serum was collected
and preserved at -20◦C until used. Whole blood samples for leukocyte count and buffy coat
isolation were used within 24 hours of sample collection.
Leukocyte and serological assay
Leukocytes were counted as described before (23). Briefly, blood samples with anticoagulant
were analyzed for total leukocyte count using an automated method. Lymphocyte, monocyte,
basophil, neutrophil and eosinophil percentages and estimated total leukocyte were determined in
blood smears stained with Giemsa.
To isolate leukocytes, the samples were centrifuged at 1500 g for 15 min, the buffy coat layer
was isolated and four volumes of 0.2% NaCl were added to induce hemolysis of red blood cells.
The product was washed 3 times with phosphate-buffered saline (PBS) and centrifuge at 200 g
for 5 min to yield leukocytes. The leukocytes were stored at -70◦C until DNA extraction.
6
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
All serum samples were analyzed using a blocking enzyme-linked immune sorbent assay
(ELISA) kit (ELISA Leukosis Blocking/BLV gp51 antibody test kit; Institut Pourquier,
Montpellier, France), according to the manufacturer’s instructions.
BLV seropositive samples that had a lymphocyte count of greater than 9,000 cells/μl were
considered to have PL(2).
Cell culture
The BLV-producing cell line, FLK (fetal lamb kidney)(25) was maintained in Dulbecco's
modified Eagle's medium (Gibco BRL) with 100 μg/ml streptomycin (Pfizer), 100 U/ml
penicillin, 50 U/ml polymyxin B, 2 mM L-glutamine, 10 μg/ml insulin, and 10% fetal bovine
serum (Sigma Chemical Co.). Cells were grown at 37 °C in a moist atmosphere containing 5%
CO2 and passaged upon reaching confluence.
DNA isolation and PCR amplification
DNA samples were extracted from leukocytes of BLV infected cattle and FLK cells (as a
control), using the Invitek (Invisorb®Spin Tissue Mini kit), according to the manufacturer’s
instructions. Standard precautions were exercised to prevent cross-contamination, including no
DNA extraction or preparation of reaction mixture in the laboratory rooms used for post-PCR
amplification work. The DNA quality and concentrations were determined using
a Picodrop spectrophotometer (Ltd Cambridge, UK) and Agarose gel electrophoresis.
In order to design primers that recognized all strains that were sampled, all existing sequences of
env gene and pX region of BLV available on the NCBI were downloaded and aligned (using the
MEGA 5 program) and primers were designed for three amplicons (env: for partial env gene,
PX1: for partial rex and tax genes, PX2: for partial tax gene and LTR region) using the MEGA 5
7
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
program. The specificity of the primers was checked by BLAST in NCBI. Sequences of primers
are shown in Table 2.
PCR was performed with a 25μl reaction mixture containing 1μl of the extracted DNA as
template, 15 mM Tris-HCl (pH 8), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide
triphosphates, 10 pM of each primer and 1.5 U Taq polymerase (YTA PCR Master Mix, Iran).
Amplification was carried out by initially denaturation at 94 °C for 3 min, followed by 35 cycles
(Table 3) and the final extension step at 72 °C for 7 min. Each batch included distilled water as a
negative control and FLK DNA as a positive control. The PCR products were electrophoresed on
a 1.5% agarose gel with the 100-bp DNA ladder (Sinaclon, Iran), stained with ethidium bromide
and visualized by ultraviolet transillumination.
Nucleotide sequencing and analysis
PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany)
and then sequenced directly in both directions in DNA Sequencing Lab of Pasteur Institute of
Iran using Genetic Analyzer 3130 (ABI, USA). The sequencing primers were the same as the
primers used for the PCR.
Each forward and reverse sequences was assembled by comparison with reference sequence
(GenBank accession number NC_001414). The consensus sequences of each samples were
submitted to GenBank (accession no. MG204538- MG204561) and aligned against the reference
and at least two sequences of each genotype, shown in Table 4, using the Clustal W algorithm of
MEGA version. Nucleotide and amino acid sequence pair distances were calculated and
phylogenetic trees were constructed based on the nucleotide sequences by the maximum
likelihood method with 1,000 bootstrap resamplings using the MEGA 7 software program.
8
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
Statistical analysis
Animal prevalence (the proportion of positive animals among the tested animals), herd
prevalence (the proportion of farms with one or more positive animals among tested herds) and
provinces prevalence (the proportion of positive animals in each seropositive province) were
statistically examined and 95% confident limits were also calculated. Student's t-test was used to
compare the specific characteristics of the hematological profile between seropositive and
seronegative cattle and to determine any significant difference in nucleotide and amino acid
sequence pair distances between clinical stages of BLV. Data are presented as mean±SEM. For
all analyses, a value of P< 0.05 was considered significant.
Results
Serological and Hematological studies
Among 280 cattle sampled, 92 (32.8%, 95% confidence interval [C.I]: 27.6_ 38.5%) were
seropositive for BLV (Table1). Seropositive animals were found in 8 of 10 provinces of Iran.
The infection rates in these areas were 0–100%, including Yazd 0%(95% C.I: 0_13% ); Markazi
53.3% (95% C.I: 36_69%); Qom 57% (95% C.I: 25_84%); Alborz 45% (95% C.I: 32.6_58.5%);
Tehran 88.8% (95% C.I: 71_97%); Razavi Khorasan 2.3%(95% C.I: 0.01_13%); East Azerbaijan
50% (95% C.I: 37_70%); Khuzestan 0%(95% C.I: 0_11%); Gilan 100%(95% C.I: 68_100%)
and Ardabil 9.5%(95% C.I: 1.4_30%) (Table1 and Figure 1).
A significant increase was detected in the total leukocyte count of BLV cattle (p<0.001). The
lymphocyte count of the BLV-positive cattle was higher compared to BLV-negative cattle
(p<0.001) and therefore the neutrophil count of the BLV-positive cattle was lower than that of
the BLV-negative cattle (p<0.001). There was no significant difference in eosinophil, monocyte,
9
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
and basophil count between the BLV-positive and BLV-negative cattle. Among BLV
seropositive animals, the rate of PL was 36.9 %. None of the seronegative animals had PL
(Supplementary Table1). The PL rate was 0-54% in the seropositive areas, including Markazi
(18.7%), Qom (0%), Alborz (37.5%), Tehran (54%), Razavi Khorasan (0%), East Azerbaijan
(45.4%), Gilan (40%), and Ardabil (0%) (Table1).
Detection of the BLV genome
At least two seropositive samples founded in 8 provinces with positive samples from 3 provinces
kindly provided by Mabna Lab were used for DNA extraction and PCR amplification. The
quality of the extracted DNA from leukocytes was suitable (Supplementary Figure 1). The 474-
bp fragments of env region, the 515-bp fragments of PX1 and the 538-bp fragments of PX2 were
successfully amplified in all samples from 11 different provinces of Iran and FLK cells
(Supplementary Figure 2).
Phylogenetic analysis based on partial env gene sequences
Phylogenetic trees were constructed using the maximum likelihood method with 1,000 bootstrap
resampling. The phylogenetic tree based on partial env gene (392 nucleotides) showed that
Iranian isolates clustered in were closed to genotypes 1 and 4. This is the first report of genotype
4 in from Iran. IR-Alborz, IR-Ardebil, IR-Ghom, IR-Markazi, IR-Mashhad, IR-Shahrekord, IR-
Tabriz and IR-FLK isolates were sorted into the genotype 1 clade and IR-Esfehan, IR-Ghazvin,
IR-Gilan and IR-Tehran isolates were sorted into the genotype 4 clade (Figure 2).
To compare our strains isolates with previous strains isolates from Iran and neighboring
countries and also strains isolates from other countries which belong to genotype 4, the
phylogenetic tree of the env gene was constructed (Supplementary Figure 3). This phylogenetic
tree showed that IR-Tabriz, IR-Mashhad, IR-Markazi, IR-Ardebil and IR-Ghom isolates of this
10
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
study were identical to TR-3128 and TR-3151 isolates from Turkey, LS1 isolate from Uruguay,
an isolate from Australia (accession no. D00647) and an isolate from Japan (accession no.
K02120). In addition, it showed that our isolates, which belonged to genotype 4 were similar to
LB59 isolate of France.
Phylogenetic analysis based on PX regions
Since genotyping of BLV is based on the env gene, in order to determine the genotypes of our
isolates based on the PX region, sequences of each genotype that had both env and PX regions
were used. All complete BLV genomic sequences available in the GenBank belong to genotypes
1, 2, 4, 6, 9 and 10 among ten BLV genotypes(21). Consequently, a maximum likelihood
phylogenetic tree was constructed based on the partial PX region (1013 nucleotides) using the
sequences from isolates of this study and complete BLV genomic sequences (Figure 3). This
phylogenetic tree, like the phylogenetic tree of the env gene, indicated that the strains isolates
found this study were sorted into genotypes 1 and 4 clades. In addition two phylogenetic trees of
PX 1 and PX 2 regions separately showed the same results (data are not shown). These results
may suggest that there is not any recombinant strain in our isolates.
Nucleotide and amino acid distances
Nucleotide and amino acid sequence pair distances were calculated using the MEGA program
and P-distance method. The results showed a nucleotide divergence percentage of -3.9% (with an
overall mean distance of 1.7%) for the env gene, 0- 2.8% (with an overall mean distance of
1.1%) for the rex gene, 0 -4.7% (with an overall mean distance of 2.3%) for the tax gene, and 0-
1.9% (with an overall mean distance of 1.0%) for the LTR region between BLV isolates from
different provinces of Iran (Supplementary Table 2). The amino acid variation percentage was 0-
3.5% (with an overall mean distance of 1.0%) for the Env, 0- 6.3% (with an overall mean
11
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
distance of 2.7%) for the Rex, and 0- 4.9% (with an overall mean distance of 2.4%) for the Tax
protein between BLV from different provinces of Iran (Supplementary Table 3).
In order to determine any significant difference in nucleotide and amino acid sequence pair
distances between clinical stages (AL and PL) of BLV, the strains used in this study and (for
more investigation) other sequences of BLV obtained from the GenBank, whose clinical stage
(AL or PL) was published, were grouped according to their genotype and clinical stage. Among
11 strains of this study that were sequenced, there were 7 sequences of genotype 1 (of which 4
were PL and 3 AL) and 4 sequences of genotype 4 (all of which were PL). In addition, there
were 14 sequences of genotype 1 and AL, 6 sequences of genotype 1 and PL, 3 sequences of
genotype 3 and AL and 6 sequences of genotype 5 and AL in GenBank. Within group and
between group nucleotide and amino acid mean distance for the env, rex and tax were measured
(Supplementary Table 4). No significant differences were observed in average changes in PL and
AL groups.
12
253
254
255
256
257
258
259
260
261
262
263
264
265
266
Figure 1. Location of different areas of Iran considered in this study: (1) Yazd with 0% infection rate; (2) Markazi with 53.3% infection rate; (3) Qom with 57% infection rate ; (4) Alborz with 45% infection rate; (5) Tehran with 88.8% infection rate; (6) Razavi Khorasan with 2.3% infection rate; (7) East Azerbaijan with 50% infection rate; (8) Khuzestan with 0% infection rate; (9) Gilan with 100% infection rate and (10) Ardabil with 9.5% infection rate.
Table 1. Epidemiological data ofcows over 2 years old (n = 280) from 10 provinces of Iran in different regions and
environments, seropositivity rates of BLV, and rate of PL.
Province
Number
of
animals
sample
d
Location of sampling
(number of samples per
farm or slaughterhouse )
No. of
seropositiv
e
%
seropositiv
e
95%
Confidence
interval
(CI)
No. of Persistent
Lymphocytosis(PL
)
%PL
Yazd301 farm000_1300
Markazi301 farm1653.336_69318.7
13
267
268269270271
272
273
274
Qom71 farm45725_8400
Alborz531 farm244532.6_58.5937.5
Tehran273 farms(12, 9, 6)2488.871_971354
Razavi
Khorasan43
3 Slaughterhouses(17,
16, 10)12.30.01_130
0
East
Azerbaijan22
2 farms(16, 6)115037_705
45.4
Khuzestan372 farms(19, 18)000_1100
Gilan101 farm1010068_100440
Ardabil211 Slaughterhouse29.51.4_3000
Total2809232.827.6_38.53436.9
Table 2. Primers which used in this study.
Primer Sequence (5΄- 3΄) Amplicon size
(bp)
Amplicon
position
(base of
ref seq.
NC_001414)
Reference
env GGGTCCTTTTATGTCAATC
GGAGGAARCCGTAGAGAG
474 5020-5500 This study
PX 1 GAAARGATCGACACCACG
GGGGACATTAGGAGAGAAG
515 7161-7676 This study
PX 2 GCCCTTCTCTCCTAATGTCC 538 7655-8202 This study
14
275
276
277
278
TTCTGGTGCCGCTAACTC
Table 3. The details of cycling conditions
Denaturation Annealing Extension Number of
cyclesTemperature
(◦C)
Time Temperature
(◦C)
Time Temperature
(◦C)
Time
env 94 30s 49 30s 72 36s 35
PX 1 94 30s 50 30s 72 36s 35
PX 2 94 30s 52 30s 72 36s 35
Table 4. BLV sequences used in this study, their origin, isolate name, genotype and GenBank accession numbers.
countryIsolateaccession
number
genotypereferences
USA-NC0014141)26(
FLK-BLVM352421)13(
USIA˟EF065644
DQ412666
1)14 ,27(
USPA˟EF065656
DQ412668
1)14 ,27(
15
279
280
281
282
283
USID˟EF065641
DQ412667
1)14 ,27(
USWI˟EF065642
DQ412669
1)14 ,27(
USCA-2˟EF065648
DQ412664
3)14 ,27(
USCA-3˟EF065649
DQ412665
3)14 ,27(
Australia-D006471)19(
Japan-K021201)28(
JPAI˟EF065657
DQ412654
1)14 ,27(
JPAI-2˟EF065651
DQ975367
1)14 ,27(
JPEH-1˟EF065652
DQ412655
1)14 ,27(
JPEH-2˟EF065653
DQ412656
1)14 ,27(
JPHY˟EF0656461)14 ,27(
16
DQ412658
JPKA-1˟EF065658
DQ412659
1)14 ,27(
JPKA-2˟EF065659
DQ412660
1)14 ,27(
JPMI-1˟EF065660
DQ412661
1)14 ,27(
JPMI-2˟EF065661
DQ412662
1)14 ,27(
JPMI-3˟EF065662
DQ975366
1)14 ,27(
JPFU˟EF065650
DQ412657
3)14 ,27(
UruguayLS1HE9673011)29(
LS2HE9673021)29(
LS3HE9673031)29(
ArgentinaAL-164FJ8085742)30(
-AF2575152)31(
PL9006FJ8085912)30(
17
PL-1238FJ8085826)30(
BelgiumLB285M352404)13(
BG˟EF065638
DQ412645
4)14 ,27(
AF5035814)20(
YR2KT1228584)9(
Costa RicaCRLC-1˟EF065655
DQ412650
5)14 ,27(
CRLC-2˟EF065654
DQ412651
5)14 ,27(
CRGC˟EF065639
DQ412649
5)14 ,27(
CRAG-1˟EF065645
DQ975368
5)14 ,27(
CRLV˟EF065643
DQ412653
5)14 ,27(
Brazil151AY1853606)32(
FranceLB59M352384)13(
Korea-AY9951741)33(
18
ItaliaI2S835307)34(
IranZanjanEU2660651)24(
TehranEU2660631)24(
KurdistanEU2660621)24(
IsfahanEU2660611)24(
ShahrekordEU2660601)24(
FLKMG2045381This study
MG2045531This study
IR-AlborzMG2045391This study
MG2045501This study
IR-ArdebilMG2045401This study
MG2045511This study
IR-EsfehanMG2045414This study
MG2045524This study
IR-GhazvinMG2045424This study
MG2045544This study
IR-GhomMG2045431This study
MG2045551This study
IR-GilanMG2045444This study
19
MG2045564This study
IR-MarkaziMG2045451This study
MG2045571This study
IR-MashhadMG2045461This study
MG2045581This study
IR-ShahrekordMG2045471This study
MG2045591This study
IR-TabrizMG2045481This study
MG2045601This study
IR-TehranMG2045494This study
MG2045614This study
Russia7VJN6958807)
Lomakina,unpublished(
MKC2137JQ6757598)
Lomakina,unpublished(
MKC3511JQ6757608)
Lomakina,unpublished(
Boliviapor2LC0806649)21(
mon1LC0806599)21(
ParaguayPar91LC0806586)21(
20
Par89LC0806576)21(
MyanmarL1LC15484810)35(
S3LC15484910)35(
TurkeyTR-3808JF8947931)36(
TR-3510JF8947921)36(
TR-3452JF8947911)36(
TR-3151JF8947901)36(
TR-3128JF8947891)36(
TR-161478FJ0091781)36(
TR-161433FJ0091771)36(
TR-496LFJ0091741)36(
TR-76LFJ0091731)36(
21
284
Figure 2. Maximum likelihood phylogenetic tree based on env gene showing that Iranian isolates were sorted into
genotypes 1 and 4 clades.
22
285
286
287
Figure 3. Maximum likelihood phylogenetic tree based on PX region constructed using sequences from this study
and complete BLV genomic sequences of genotypes 1, 2, 4, 6, 9 and 10, showing that Iranian strains clustered into
genotypes 1 and 4.
23
288
289
290
291
292
Discussion
In this study 280 cattle from 10 provinces of Iran were sampled. The total prevalence of BLV
infection was 32.8% (95% C.I: 27.6_ 38.5%) and infection rates ranged from 0% to 100% in the
sampled areas (Table1). The prevalence can be related to management or sanitary practices. The
high density of animals on a farm where infected and uninfected animals are in continuous
contact or multiple uses of injection needles during vaccinations or treatments and also the same
gloves and sleeves for rectal palpation may contribute to the infection transmission in the
herd(37). Furthermore, the presence of vector insects and climatic conditions can affect the
prevalence (38, 39). Comparison of the results with previous studies in Iran, showed that the
prevalence of the infection has increased in some provinces such as Tehran, Alborz, Markazi,
East Azerbaijan and Ardabil (40-43). It may be a consequence of increased industrial cattle in
Iran in recent years and lack of established control programs for BLV in our farms. Our finding
also showed no seroprevalence (95% C.I: 0_11%) in Khuzestan. Similar prevalence rates of
BLV infection were reported from Khuzestan, including 0% in 1996(40) and 0.5% in 2005(44)
indicating a low prevalence of BLV infection in Khuzestan area compared to other provinces. It
may due to the smaller number of industrial herds in Khuzestan or its hot climate. Totally, our
findings confirmed a higher prevalence of BLV infection in industrial herds compared to less
industrial herds.
Our results revealed that seropositive cattle had higher leukocyte ands lymphocyte count and a
lower neutrophil count than seronegative cattle (P<0.001). As in previous studies(2), the rate of
PL in this study was approximately 30%.
Analysis of the BLV env gene sequences of isolates from different geographical areas showed
that BLV strains were classified into ten genotypes and those in the same clade had a common
origin(21). There are a few phylogenetic studies of partial BLV env and gag sequences in limited
24
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
provinces of Iran(24, 45). This study was performed on partial sequences of env, rex and tax
genes and LTR region of BLV isolates from 13 province of Iran, making it the first Iranian large
study of the partial sequences of the rex and tax genes and LTR region. Our results showed that
Iranian isolates were sorted into genotypes 1 and 4 clades, which is the first report of genotype 4
from in Iran. It may due to examination of more samples and designing primers that could
recognize all strains in our samples. If genotyping of isolates is performed based on partial
sequences of one gene, it is probable that the isolates are intersubtype recombinants and may
belong to another genotype according to another gene(46). Since phylogenetic trees of env, rex
and tax genes and LTR region indicated the same results that isolates of this study were sorted
into genotypes 1 and 4 clades, there may not be any recombinant strains in our strains isolates.
As shown in Figures 2 and 3 and Suppl. Fig.3, phylogenetic analysis of env gene indicated that
IR-Tabriz, IR-Mashhad, IR-Markazi, IR-Ardebil and IR-Ghom strains from isolates of this study
which belonged to genotype 1, were identical to TR-3128 and TR-3151 isolates from Turkey, an
isolate from Australia (accession no. D00647), LS1 isolate from Uruguay (accession no.
HE967301) and an isolate from Japan (accession no. K02120). These results suggest that these
isolates have a common source; however, based on the PX region, they had a slight distance,
which may be due to the fact that the env gene of these strains are isolatesis more conserved than
the PX region.
Hemmatzadeh (2007)(24) found that five Iranian strains isolates of Iran were sorted into
genotype 1 clade near the strain isolate from Australia (accession no. D00647) and far from an
strain isolate from Korea (accession no. AY995174). As mentioned previously, similar results
were obtained in our study. However, phylogenetic analysis of the gag gene of a strain an isolate
from Iran by Momtaz (2010) (47) indicated that the Iranian strain isolate was similar to an
American strain isolate (accession no. NC001414) and different from the Australian strain isolate
25
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
(accession no. D00647). Nonetheless, this distinct difference may be due to using similar stains
in that phylogenetic study.
Our results showed that four strains belonged to genotype 4 and phylogenetic tree of these
sequences with other isolates of genotype 4 sequences from other countries demonstrated that
our strains were similar to the LB59 from France (accession no. M35238). Although according to
Hemmatzadeh (2007), the LB59 was sorted into genotype 1 clade with the FLK isolate, Balic et
al. (2012)(48) and our study showed that it belonged to genotype 4. Since in the study by
Hemmatzadeh (2007), Iranian strains belonged to genotype 1, they were sorted at a great
distance from a Belgian isolate (accession no. AF503581) that belonged to genotype 4.
Nevertheless, our Iranian strains, which belonged to genotype 4 were sorted near to the Belgian
strain.
To conclude, our results showed that Iranian strains that belonged to genotype 1 were similar to
isolates from Australia, Japan, Uruguay and Turkey and those that belonged to genotype 4 were
similar to strains from France and Belgium. Iran has been importing biological products and
cows from other countries for many years; therefore, the similarity between Iranian strains and
strains from those countries may be a reason for the common origin. Nucleotide and amino acid
sequence pair distances showed no difference between IR-Markazi and IR-Ghom strains in env,
rex and tax genes and LTR region. Since these strains were from two neighboring provinces, it is
likely that there is a strain in circulation in these two provinces.
Among our strains, the overall mean distance of nucleotide divergence percentage for env, rex
and tax genes and LTR region was 1.7%, 1.1%, 2.3% and 1.0% respectively. This diversity only
consisted of nucleotide substitution and there was no frame shift by insertion or deletion
mutations. The overall mean distance of amino acid variation percentage for Env, Rex and Tax
proteins was 1.0%, 2.7% and 2.4% respectively. However, some studies found a very low rate of
26
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
mutation in the env gene as a highly conserved region(19, 20). According to our study and more
recent studies, the env gene is more variable than previously thought(14, 34). Mcgirr and
Buehuring (2006)(49) found that the maximum changes were 5% and 7% in nucleotide
sequences of rex and tax genes respectively and 11% and 9% in amino acid sequences
respectively. Our results support their hypothesis that the Rex protein can withstand more amino
acid changes compared to the Tax protein, suggesting that the Tax protein experiences higher
evolutionary constraints and is more conserved. Zhao et al. (2007)(27) analyzed the pXBL
region and reported that the overall mean distance of nucleotide and amino acid divergence
percentage was 1.86% and 2.12% for tax, 1. 4% and 3.38% for rex, 1.24% and 3.73% for R3 and
1.29% and 3.18% for G4 respectively. They proposed that Tax protein was the most conserved
among the four regulatory proteins, indicating its importance in the viral life cycle and the
process of evolution and natural selection. The results of our study as well as a previous study
(50) showed no significant difference in nucleotide and amino acid sequence pair distances
between clinical stages (AL and PL) of BLV.
Conclusion
In conclusion, the results of this study showed a high seroprevalence of BLV infection in Iran.
Moreover, the findings implied that not only BLV is a cattle health problem in Iran, but also the
infected population is growing, which has many economic consequences. For the first time, our
investigation using phylogenetic analysis of env, rex and tax genes and LTR region of BLV
revealed that two different BLV genotypes (1 and 4) were present in Iran. Our results support
previous studies indicating that the env gene is more variable than previously thought, the Rex
protein can withstand more amino acid changes compared to the Tax protein and there is no
significant difference in nucleotide and amino acid sequence pair distances between clinical
27
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
stages. These results provide important information to enable the implementation of appropriate
cattle-management policies in order to develop more-effective methods of BLV limitation in
Iran.
Conflicts of interest:
There are no conflicts of interest.
Acknowledgements:
We thank Mr. Mehdi Kamyabi, Dr. Alireza Koochakzadeh, Dr. Mohammad Khosravi, Dr.
Alireza Tarahi, Dr. Samad Muhammadnejad, Dr. Babak shafiee, Dr. Jamaluddin Al Masum, Dr.
Siamak Rezvan, Dr. Mohammad Tooloei, Dr. Samad Lotfollahzadeh, and Mabna lab for kindly
assisting with the large-scale sampling from many farms in Iran. This work was supported by
Grants-in-Aid for Scientific Research in Pasteur Institute of Iran and Veterinary Medicine of
University of Tehran.
ReferencesPrimary Sources
Secondary Sources
Uncategorized References
1. Maclachlan NJ, Dubovi EJ, Fenner F. Fenner's veterinary virology. Amsterdam; Boston: Elsevier Academic Press, 264-266; 2011.2. Otto M. Radostits CCG, Kenneth W. Hinchcliff, Peter D. Constable, editor. Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats. 10th ed: ELSEVER; 2007.3. Tsutsui T, Kobayashi S, Hayama Y, Nishiguchi A, Kameyama K, Konishi M, et al. Estimation of the within-herd transmission parameter of bovine leukemia virus. Preventive veterinary medicine. 2010;95(1-2):158-62.4. Polat M, Takeshima S-n, Aida Y. Epidemiology and genetic diversity of bovine leukemia virus. Virology journal. 2017;14(1):209.5. Kobayashi S, Tsutsui T, Yamamoto T, Hayama Y, Kameyama K, Konishi M, et al. Risk factors associated with within-herd transmission of bovine leukemia virus on dairy farms in Japan. BMC veterinary research. 2010;6(1):1.
28
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404405406407408409410411412413414415
6. Lassauzet M, Thurmond M, Johnson W, Holmberg C. Factors associated with in utero or periparturient transmission of bovine leukemia virus in calves on a California dairy. Canadian journal of veterinary research. 1991;55(3):264.7. Johnson R, Kaneene JB. Bovine leukemia virus and enzootic bovine leukosis. Vet Bull. 1992;62(4):287-312.8. Van Driessche B, Rodari A, Delacourt N, Fauquenoy S, Vanhulle C, Burny A, et al. Characterization of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome. Scientific reports. 2016;6:31125.9. Durkin K, Rosewick N, Artesi M, Hahaut V, Griebel P, Arsic N, et al. Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs. Retrovirology. 2016;13(1):33.10. Safari R, Hamaidia M, de Brogniez A, Gillet N, Willems L. Cis-drivers and trans-drivers of bovine leukemia virus oncogenesis. Current opinion in virology. 2017;26:15-9.11. Bangham CR. Human T cell leukemia virus type 1: persistence and pathogenesis. Annual review of immunology. 2018;36:43-71.12. Rosewick N, Durkin K, Artesi M, Marcais A, Hahaut V, Griebel P, et al. Cis-perturbation of cancer drivers by the HTLV-1/BLV proviruses is an early determinant of leukemogenesis. Nature communications. 2017;8:15264.13. Mamoun R, Morisson M, Rebeyrotte N, Busetta B, Couez D, Kettmann R, et al. Sequence variability of bovine leukemia virus env gene and its relevance to the structure and antigenicity of the glycoproteins. Journal of virology. 1990;64(9):4180-8.14. Zhao X, Buehring GC. Natural genetic variations in bovine leukemia virus< i> envelope</i> gene: Possible effects of selection and escape. Virology. 2007;366(1):150-65.15. Moratorio G, Fischer S, Bianchi S, Tomé L, Rama G, Obal G, et al. A detailed molecular analysis of complete bovine leukemia virus genomes isolated from B-cell lymphosarcomas. Veterinary research. 2013;44(1):19.16. Ayral A-M, Clarkson S, Cheeseman M, Wells S, Dear TN. A panel of optimized primers and positive-control DNAs for PCR detection of common biological contaminants in mouse cell lines and tissue samples. Lab Animal. 2006;35:31.17. McGirr K, Buehring G. tax and rex Sequences of bovine leukaemia virus from globally diverse isolates: rex amino acid sequence more variable than tax. Journal of Veterinary Medicine, Series B. 2005;52(1):8-16.18. Choi E-A, Hope TJ. Mutational analysis of bovine leukemia virus Rex: identification of a dominant-negative inhibitor. Journal of virology. 2005;79(11):7172-81.19. Coulston J, Naif H, Brandon R, Kumar S, Khan S, Daniel R, et al. Molecular cloning and sequencing of an Australian isolate of proviral bovine leukaemia virus DNA: comparison with other isolates. Journal of General Virology. 1990;71(8):1737-46.20. Willems L, Thienpont E, Kerkhofs P, Burny A, Mammerickx M, Kettmann R. Bovine leukemia virus, an animal model for the study of intrastrain variability. Journal of virology. 1993;67(2):1086-9.21. Polat M, Takeshima S-n, Hosomichi K, Kim J, Miyasaka T, Yamada K, et al. A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis. Retrovirology. 2016;13(1):4.22. Gregory L, Gaeta NC, Araújo J, Thomazelli LM, Harakawa R, Ikuno AA, et al. Bovine leukaemia virus genotypes 5 and 6 are circulating in cattle from the state of São Paulo, Brazil. Journal of Medical Microbiology. 2017;66(12):1790-7.23. Kazemimanesh M, Madadgar O, Mahzoonieh M, Zahraei-Salehi T, Steinbach F. A Serological Study on Bovine Leukemia Virus Infection in Some Provinces of Iran between 2010 and 2012. Iranian Journal of virology. 2013;6(3):1-7.
29
416417418419420421422423424425426427428429430431432433434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463
24. Hemmatzadeh F. Sequencing and phylogenetic analysis of gp51 gene of bovine leukaemia virus in Iranian isolates. Veterinary research communications. 2007;31(6):783-9.25. Van der Maaten M, Miller J. Replication of bovine leukemia virus in monolayer cell cultures. 1976.26. Petropoulos C. Retroviral taxonomy, protein structures, sequences, and genetic maps. Retroviruses Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1997:757-805.27. Zhao X, McGirr KM, Buehring GC. Potential evolutionary influences on overlapping reading frames in the bovine leukemia virus pXBL region. Genomics. 2007;89(4):502-11.28. Sagata N, Yasunaga T, Tsuzuku-Kawamura J, Ohishi K, Ogawa Y, Ikawa Y. Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses. Proceedings of the National Academy of Sciences. 1985;82(3):677-81.29. Moratorio G, Obal G, Dubra A, Correa A, Bianchi S, Buschiazzo A, et al. Phylogenetic analysis of bovine leukemia viruses isolated in South America reveals diversification in seven distinct genotypes. Archives of virology. 2010;155(4):481-9.30. Rodriguez SM, Golemba MD, Campos RH, Trono K, Jones LR. Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades. Journal of General Virology. 2009;90(11):2788-97.31. Dube S, Dolcini G, Abbott L, Mehta S, Dube D, Gutierrez S, et al. The complete genomic sequence of a BLV strain from a Holstein cow from Argentina. Virology. 2000;277(2):379-86.32. Camargos M, Stancek D, Rocha M, Lessa L, Reis J, Leite R. Partial sequencing of env gene of bovine leukaemia virus from Brazilian samples and phylogenetic analysis. Journal of Veterinary Medicine, Series B. 2002;49(7):325-31.33. Lim SI, Jeong W, Tark DS, Yang DK, Kweon CH. Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-). Journal of Veterinary Science. 2009;10(4):331-6.34. Molteni E, Agresti A, Meneveri R, Marozzi A, Malcovati M, Bonizzi L, et al. Molecular characterization of a variant of proviral bovine leukaemia virus (BLV). Journal of Veterinary Medicine, Series B. 1996;43(1 10):201-11.‐35. Polat M, Moe HH, Shimogiri T, Moe KK, Takeshima S-n, Aida Y. The molecular epidemiological study of bovine leukemia virus infection in Myanmar cattle. Archives of virology. 2017;162(2):425-37.36. Alkan F, Oğuzoğlu TÇ, Timurkan MO, Karapınar Z. Characterisation of env and gag gene fragments of bovine leukemia viruses (BLVs) from cattle in Turkey. Archives of virology. 2011;156(10):1891-6.37. Gutierrez G, Alvarez I, Politzki R, Lomonaco M, Santos MJD, Rondelli F, et al. Natural progression of Bovine Leukemia Virus infection in Argentinean dairy cattle. Veterinary microbiology. 2011;151(3-4):255-63.38. Zaghawa A, Beier D, Abd El-Rahim IH, Karim I, El-ballal S, Conraths FJ, et al. An outbreak of enzootic bovine leukosis in upper Egypt: clinical, laboratory and molecular-epidemiological studies. J Vet Med B Infect Dis Vet Public Health. 2002;49(3):123-9.39. Murakami K, Kobayashi S, Konishi M, Kameyama K, Yamamoto T, Tsutsui T. The recent prevalence of bovine leukemia virus (BLV) infection among Japanese cattle. Veterinary microbiology. 2011;148(1):84-8.40. Kargar R, Hessami M, Ahourai P, Ghabosi B, khedmati K, Ezzi A, et al. Seroepidemiological study of Enzootic Bovine Leukosis in Iran. 1996;9(30):164-6.41. Tooloei M, Bazargani TT, Broujeni GN, Khaki Z, Bokaei S, Ashrafei I. An abattoir surveyon prevalence of Enzootic bovine leukosis virus infection and associated clinical, haematologicaland flowcytometric findings in holestein cattle in tehran. JVetRes. 2009;64(2):147-56.
30
464465466467468469470471472473474475476477478479480481482483484485486487488489490491492493494495496497498499500501502503504505506507508509510
42. Nikbakht Brujeni G, Poorbazargani TT, Nadin-Davis S, Tolooie M, Barjesteh N. Bovine immunodeficiency virus and bovine leukemia virus and their mixed infection in Iranian Holstein cattle. The Journal of Infection in Developing Countries. 2010;4(09):576-9.43. Ghaemmaghami S, oliai MM, H N, Firouzi M, Bakhshesh M. Serologica survey of bovine enzootic leukosis in Markazi Province. 1999;54(1):11-3.44. Haji Hajikolaei MR, Sayfiabad shapouri MR, Akbari M. Serological study of Bovine Leukemia Virus (BLV) infection in cattle in Ahwaz. Pajouhesg & sazandegi. 2005;71:26-30.45. Momtaz H. Cloning and phylogenetic analysis of bovine leukemia virus gag gene in Iranian isolate. African Journal of Microbiology Research. 2010;4(3):218-21.46. Desrames A, Cassar O, Gout O, Hermine O, Taylor GP, Afonso PV, et al. Northern African strains of human T-lymphotropic virus type 1 arose from a recombination event. Journal of virology. 2014;88(17):9782-8.47. Momtaz H, Hemmatzadeh F. A Serological survey of Bovine Leukemia Virus of cattle in Chaharmahal Bakhtiary province of Iran. Iranian Journal of Veterinary Research. 2003;4(1):37-44.48. Balić D, Lojkić I, Periškić M, Bedeković T, Jungić A, Lemo N, et al. Identification of a new genotype of bovine leukemia virus. Archives of virology. 2012;157(7):1281-90.49. McGirr KM, Buehuring GC. Tax & rex: overlapping genes of the Deltaretrovirus group. Virus genes. 2006;32(3):229-39.50. Panei CJ, Serena MS, Metz GE, Bravi ME, Echeverría MG, González ET. Analysis of the pX region of bovine leukemia virus in different clinical stages of Enzootic Bovine Leukemia in Argentine Holstein cattle. Virus research. 2012.
31
511512513514515516517518519520521522523524525526527528529530531
532
