GENETIC IDENTIFICATION OF BOVINE LEUKAEMIA VIRUS
Abstract and keywords
Abstract (English):
Molecular genetic research methods make it possible to evaluate the genetic diversity of bovine leukemia virus (BLV) and are the most informative approaches to its genetic identification. Molecular genetic research methods work well for the phylogenetic analysis of sequenced nucleotide DNA sequences of the provirus, as well as for the polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) according to the phylogenetic classification of the pathogen. The purpose of the research was to study the scientific and methodological approaches to the genetic identification of bovine leukemia virus, integrated into the molecular monitoring of infection of cattle with BLV genotypes. The authors used PCR-RFLP-genotyping and comparative phylogenetic analysis of aligned nucleotide sequences of the env gene fragment of the BLV provirus isolates to detect the genotypic affiliation of the cattle from twenty-one livestock farms of the Republic of Tatarstan. As a result, isolates of four out of ten BLV genotypes were found in the Tatarstani cattle, namely genotypes 1, 4, 7, and 8. The research involved a comparative analysis of 505 nucleotide sequences of a fragment of the BLV env gene, including those deposited in GenBank NCBI. The analysis confirms the inconsistency of several earlier PCR-RFLP typing strategies with the current approach in assessing the genotypic diversity by phylogenetic analysis. The improved strategy of PCR-RFLP genotyping of BLV corresponds with its modern phylogenetic classification. The strategy makes it possible to identify all the known genotypes of the viral pathogen. Its validity has been proved by in silico modelling of restrictogrammes and a phylogenetic analysis of the env gene fragment of 57 reference isolates of ten BLV genotypes that generate 57 genotype-associated combinations of diagnostically significant PCR-RFLP profiles.

Keywords:
Bovine leukaemia virus, BLV, cattle, gene, genotype, genetic identification, PCR, RFLP, sequencing
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INTRODUCTION

Enzootic Bovine Leukosis (EBL) is a chronic infectious disease of a tumorous nature. It causes significant economic damage to the dairy and beef cattle industry due to poor production, low quality, cattle mortality, and expensive epidemic prevention measures [1, 2].

Foodstuffs of infected animals can be dangerous to humans due to the harmful metabolites it contains. The causative agent affects all kinds of raw material (milk, meat, by-products) and products, remaining a potential source of human infection [35].

Pasteurization of milk inactivates the virus but does not degrade its genome. The genetic material of the provirus maintains its integrity in canned meat [6]. Moreover, there are dairy products with partial pasteurization regime, e.g. classic cheeses, granulated cottage cheese, powdered milk with a low heating temperature, etc. The temperature processing parameters used in accordance with the regulatory and technical documentation cannot destroy harmful metabolites and, in some cases, do not kill the virus [7].

According to some researches, DNA of BLV provirus was found in epithelial cells of human mammary glands, including those of breast cancer patients. The hypothesis states that BLV may destabilize the host genome, thus leading to cancerous degeneration of cells [812].

Obtaining high-quality raw materials of animal origin is the most important challenge for meat and dairy industry. The challenge includes the development of functional and gerodietic foods [1315].

According to the requirements of the Technical Regulations of the Customs Union On safety of milk and dairy products (TR CU 033/2013), BLV preventive measures and eradication activities are extremely important, given the significant prevalence of this incurable disease in the Russian Federation [16, 17].

An early genetic diagnosing of the pathogen is part of the system of anti-epizootic measures, followed by the removal of infected animals from the herd.  Molecular genetic research methods make it possible to assess the genetic diversity of BLV [18]. This is the most informative approach to the gene identification of the virus. Molecular genetic research methods work well for the phylogenetic analysis of nucleotide DNA sequences of the provirus, as well as for PCR-RFLP analysis according to the phylogenetic classification of the pathogen [19].

The current phylogenetic classification of BLV includes ten genotypes. The first seven genotypes were described by Argentinean scientists in 2009 [20], while genotype 8 was described by researchers from Russia [2123], Croatia [24], and a European team of scientists [25] in 20112013. Genotype 9 was investigated by a team of Argentinean, Chilean, and Japanese scientists in 2016 [26]. Genotype 10 was described by a team of researchers from Thailand and South Korea [27] in 2016.

The objective of the current research was to study the scientific and methodological approaches to the genetic identification of BLV integrated into the molecular monitoring of infection of cattle herds with BLV genotypes. The following tasks were set:

  • to establish the genotypes of BLV isolates in Tearstain cattle;
  • to define the types of BLV isolates with deciphered nucleotide sequences of the env gene fragment, depending on the chosen gene identification strategy;
  • to improve the strategy of PCR-RFLP-genotyping of BLV and make it consistent with the modern phylogenetic BLV classification.

 

STAGY OBYECTS AND METHODS

The research involved a total of 179 blood samples from AGID-positive cows. The samples were provided by agricultural enterprises from 21 districts of the Republic of Tatarstan. The samples were genetically examined for BLV. The examination included a phylogenetic analysis of sequenced env gene fragment of the pathogen and a PCR-RFLP-genotyping consistent with the phylogenetic classification of the infectious agent.

To extract DNA from the whole conserved blood, we used a commercial PCR diagnostic kit, ‘DNA-Sorb B’, produced by the Central Research Institute of Epidemiology of the Federal Supervisory Service for Consumer Rights and Human Welfare, Ministry of Health of the Russian Federation.

Nested PCR with extracted samples of BLV proviral DNA was performed with external (env5032 and env5608) and internal (env5099 and env5521) primers initiating the generation of env gene 444 bp fragment of causative agent at the final stage of reaction [28].

Restriction endonucleases used in PCR-RFLP-genotyping of BLV were consistent with its phylogenetic classification: BstYI (isoshizomer BstX2I), HphI (isoshizomer AsuHPI), HaeIII, PvuII, SspI. The NEBcutter v.2.0 web resource was used for PCR-RFLP modelling.

For the detection of the obtained results of PCR and PCR-RFLP analysis, 2.5% agarose gel horizontal electrophoresis was applied with a TBE buffer (pH 8.0) containing ethidium bromide. The electrophoregrammes were examined in a UV-transilluminator (λ = 310 nm). The sizes of the generated DNA fragments were compared with standard DNA molecular weight markers (SibEnzyme Ltd, Russia).

Sequencing of the PCR amplification products of the env gene fragment of detected BLV provirus isolates was performed on ABI PRISM 3100 Genetic Analyser (Applied Biosystems, USA) in the laboratory of Scientific and Technical Complex Sintol (Russia). Internal oligonucleotide primers env5099 and env5521 were used as sequencing. The sequenced fragments of the env gene of BLV provirus isolates were aligned with the corresponding nucleotide sequences of the reference BLV isolates from GenBank with the help of BLAST and MEGA-4 programmes. The last stage included a phylogenetic analysis.

 

RESULTS AND DISCUSSION

The study featured a PCR-RFLP-genotyping and a comparative phylogenetic analysis of the aligned sequences of the env gene fragment of BLV provirus isolates from 21 districts of the Republic of Tatarstan.

As a result, out of 179 identified isolates, ten isolates belonged to genotype 1; 106 isolates belonged to the cluster of genotype 4; 55 were characterized as genotype 7, and the remaining eight provirus isolates belonged to genotype 8 (Table 1).

According to the results obtained by PCR-RFLP-genotyping and phylogenetic analysis of sequenced env gene fragment, there are four out of ten currently known BLV genotypes in Tatarstan: 1, 4, 7, and 8.

Fig. 1 shows the genotypes of BLV isolated with the help of phylogenetic analysis of nucleotide sequences of env gene fragment.

An additional assessment of the heterogeneity of the reference BLV representatives for the env gene included an analysis of the intra- and intergenotypic heterogeneity of genotypes. The data in Table 2 indicate that it is impossible to use the ‘heterogeneous’ criterion for assessing the genetic diversity of BLV.

As part of the next task, BLV isolates with the decoded nucleotide sequences of the env gene fragment were identified according to the chosen genetic identification strategy. The degree of consistency of genotypic approaches was assessed by comparing the data of the in silico PCR-RFLP and the phylogenetic analyses.

A comparative analysis of 505 nucleotide sequences of the BLV env gene locus, including those deposited with GenBank NCBI, confirms the inconsistency of a number of earlier PCR-RFLP typing strategies [28–30] with the current approach in assessing the genotypic diversity by means of phylogenetic analysis.

Thus, the BLV isolates that belong to the Belgian subgroup according to D. Beier et al. (2001) [28], belong to genotype 4 according to the phylogenetic classification; Australian subgroup can be referred to genotypes 1, 3, 6, 8, or 9; Japanese subgroup – to genotypes 1, 6, or 7. In addition, the genotyping strategy [28] includes 11 additional unique combinations of PCR-RFLP profiles, conditionally identical to 11 unclassifiable BLV subgroups (Table 3).

Besides, BLV isolates that belong to genotype 1 and 7 according to the phylogenetic analysis, can be referred to Australian and Japanese subgroups, as well as to three unclassifiable subgroups, according to the strategy of D. Beier et al. (2001) [28]; genotypes 2, 5, and 10 belong to two unclassifiable subgroups; genotypes 3 and 8 – to Australian subgroup; genotype 4 – to the Belgian subgroup and four unclassifiable subgroups; the genotype 5 – to two unclassifiable subgroups; genotype 6 – to the Australian, Japanese and two unclassifiable subgroups; genotype 9 – to the Australian and one unclassifiable subset (Table 3).

BLV isolates that were genotyped according to
M. Licursi et al. (2002) [29] as genotype 1 may belong to genotypes 1, 4, 6, or 7 according to the phylogenetic classification; genotype 3 – to genotypes 1, 6, or 7; genotype 5 – to genotypes 1, 3, 6, 7, or 9; genotype 6 – to genotypes 2, 4, 5, or 7 (Table 4).

For the genotyping strategy described in [29], there are 19 unique combinations of PCR-RFLP profiles that are conditionally identical to 19 unclassifiable BLV genotypes (Table 4).

Besides, BLV isolates that are genotyped according to phylogenetic analysis as genotype 1 may refer to 1, 3, 5, and three unclassifiable BLV genotypes according to the strategy of M. Licursi et al. (2002) [29]; genotype 2 belongs to genotype 6 and two unclassifiable genotypes; the genotype 3 – to genotype 5 and one unclassifiable genotype; genotype 4 – to genotypes 1 and 6 and five unclassifiable genotypes; genotype 5 – to genotype 6 and two unclassifiable genotypes; genotype 6 – to genotypes 1, 3, and 5 and three unclassifiable genotypes; genotype 7 – to genotypes 1, 3, and 6 and four unclassifiable genotypes; genotype 8 – to one unclassifiable genotype; genotype 9 – to genotype 5; genotype 10 – to three unclassifiable genotypes (Table 4).

It should be mentioned that, when analyzing in silico PCR-RFLP data from 505 BLV representatives, we found not a single nucleotide sequence of the env gene fragment that would belong to genotypes 2 and 4 according to M. Licursi et al. (2002) (Table 4). This fact did not make it possible to prove the actual existence of PCR-RFLP profiles indicated for these two BLV genotypes.

 

 

Table 1. Distribution of 179 genotyped samples of BLV provirus DNA according to 21 districts of the Republic of Tatarstan, Russian Federation

 

Districts

Number of analysed samples

BLV genotypes

1

2

3

4

5

6

7

8

9

10

1

Aznakaevsky

10

9

1

2

Al’keyevsky

13

5

8

3

Arsky

7

6

1

4

Buinsky

7

2

3

2

5

Vysogorsky

4

4

6

Drozhanovsky

12

4

7

1

7

Zainsky

8

7

1

8

Klaibitsky

7

7

9

Laishevsky

13

12

1

10

Leninogorsky

19

15

4

11

Mamadyshksy

10

10

12

Menzelinsky

6

6

13

Musl’umovsky

4

4

14

Nizhnekamensky

14

8

5

1

15

Pestrechinsky

1

1

16

Rybnoslobodsky

8

8

17

Sarmanovsky

2

2

18

Spassky

9

3

6

19

Tukaevsky

9

4

3

2

20

T’ulyachinsky

6

6

21

Chistopolsky

10

3

6

1

Total number of samples

179

10

106

 

55

8

Fig. 1. Dendrogramme of 99 isolates of 10 BLV genotypes, based on a phylogenetic analysis of the env gene fragment [MEGA-4: algorithm NJ, 400 nt, 99 seq.] Legend: black diamond marks GenBank NCBI nucleotide sequences of the env gene fragment of BLV provirus isolates in the Republic of Tatarstan.

 

Table 2. Intra- and intergenotypic heterogeneity of reference BLV representatives according to env gene (% ratio)

 

PHYLOGENETIC CLASSIFICATION OF BLV

GENOTYPE

1

2

3

4

5

6

7

8

9

10

1

0–5

3–6

3–7

3–7

3–7

3–7

3–8

2–6

3–6

4–7

2

3–6

0–1

3–4

2–4

4–5

3–5

3–5

2–4

3

4–6

3

3–7

3–4

0–2

3–5

4–5

3–5

3–6

3–4

3

4–6

4

3–7

2–4

3–5

0–3

3–5

2–5

2–6

2–4

2–4

3–5

5

3–7

4–5

4–5

3–5

0–2

4–6

3–5

4–5

4–5

5–6

6

3–7

3–5

3–5

2–5

4–6

0–4

3–6

2–5

3–5

3–5

7

3–8

3–5

3–6

2–6

3–5

3–6

0–4

2–5

3–5

4–6

8

2–6

2–4

3–4

2–4

4–5

2–5

2–5

0–2

2–3

3–5

9

3–6

3

3

2–4

4–5

3–5

3–5

2–3

0–1

4–5

10

4–7

4–6

4–6

3–5

5–6

3–5

4–6

3–5

4–5

0–2

 

Table 3. Comparison of in silico data for PCR-RFLP (typification according to D. Beier et al., 2001) and the phylogenetic analysis of the BLV env gene fragment

 

PCR-RFLP genotyping

PCR product

(bp)

RFLP fragments (bp)

BLV genotypes

N

PvuII

BamHI

BclI

1

2

3

4

5

6

7

8

9

10

Subgroup

Belgian

444

280/164

444

225/219

142

142

Australian

444

444

316/128

225/219

57

4

28

70

21

19

199

Japanese

444

444

316/128

219/121/104

8

6

1

15

?

444

444

444

225/219

43

1

2

3

17

66

?

444

444

316/128

444

1

14

2

17

?

444

444

316/128

225/191/28

1

1

?

444

280/164

316/128

225/219

36

10

3

49

?

444

280/164

316/128

444

1

1

?

444

280/164

316/128

219/189/36

1

1

?

444

280/164

444

444

1

1

?

444

208/164

253/191

225/219

1

1

?

444

280/164

444

219/121/104

4

4

?

444

444

242/128/74

225/219

1

1

?

444

444

444

444

7

7

Legend. N is the number of analysed BLV isolates with an established PCR-RFLP profile; “?” – an unclassifiable BLV taxon.

 

Table 4. Comparison of in silico data of PCR-RFLP (typification according to M. Licursi et al., 2002) and phylogenetic analysis of a fragment of the BLV env gene  

 

PCR-RFLP genotyping

PCR product

(bp)

RFLP fragments (bp)

BLV genotypes

N

BclI

HaeIII

PvuII

1

2

3

4

5

6

7

8

9

10

Genotype

1

444

225/219

198/94/87/32/27/6

444

98

1

28

68

195

2

444

219/121/104

312/94/32/6

444

3

444

219/121/104

285/94/32/27/6

444

8

6

1

15

4

444

219/121/104

198/94/87/32/27/6

444

5

444

225/219

285/94/32/27/6

444

1

3

1

1

22

28

6

444

225/219

198/94/87/32/27/6

280/164

35

139

9

3

186

?

444

444

198/94/87/32/27/6

444

1

12

7

20

?

444

225/191/28

198/119/94/27/6

444

1

1

?

444

225/219

312/94/32/6

444

1

1

?

444

444

198/94/87/32/27/6

280.164

1

1

2

?

444

219/189/36

198/94/87/32/27/6

280/164

1

1

?

444

225/219

285/94/32/27/6

280/164

2

1

3

?

444

444

198/87/49/45/32/27/6

444

1

1

?

444

225/219

225/94/87/32/6

444

21

 

21

?

444

225/219

285/94/32/21/6/6

444

1

1

?

444

225/219

198/121/87/32/6

280/164

1

1

?

444

225/219

198/119/94/27/6

280/164

1

1

?

444

219/121/104

285/94/32/27/6

280/164

4

4

?

444

225/219

198/87/49/45/32/27/6

444

2

2

?

444

225/219

198/119/94/27/6

444

1

1

?

444

444

198/121/87/32/6

444

1

1

2

?

444

225/219

198/87/49/45/32/27/6

280/164

1

1

?

444

444

198/94/81/32/21/6/6

444

1

1

?

444

225/219

198/94/81/32/27/6/6

444

6

6

?

444

225/219

279/94/32/27/6/6

444

11

11

Legend. N is the number of analysed BLV isolates with an established PCR-RFLP profile; “?” – an unclassifiable BLV taxon.

 

Table 5 compares the in silico data of PCR-RFLP according to the strategy by H. Fechner et al. (1997) [30] and the phylogenetic analysis of a fragment of the BLV env gene.

Thus, the BLV isolates identified according to H. Fechner et al. (1997) [30] as variant group A belong to genotype 4 according to the phylogenetic classification; variant group B – to genotypes 1, 6, or 7; variant group C – to genotypes 1, 3, 6, 7, or 9; variant group D – to genotypes 1, 4, or 7; variant group F – to genotypes 2, 5, or 7; variant group G – to genotype 1 (Table 5).

For this typing strategy [30], there are 17 unique combinations of PCR-RFLP profiles that are conditionally identical to 17 unclassifiable variant BLV groups (Table 5). Besides, BLV isolates genotyped by phylogenetic analysis as genotype 1 are characterized as variant groups B, C, D, G and three unclassifiable variant groups of BLV according to the strategy of H. Fechner et al. (1997) [30]; the genotype 2 belongs to variant group F and one unclassifiable variant group; genotype 3 – to variant group C; genotype 4 – to variant groups A, D and three unclassifiable variant groups; genotype 5 – to variant group F and two unclassifiable variant groups; genotype 6 – to variant groups B and C and four unclassifiable variant groups; genotype 7 – to variant groups B, C, D, F and two unclassifiable variant groups; genotype 8 – to variant group E; genotype 9 – to variant group C and one unclassifiable variant group; genotype 10 – to three unclassifiable variant groups of BLV (Table 5).

The priority task of the research was to improve the strategy of PCR-RFLP genotyping of BLV by making it consistent with the phylogenetic classification and taking into account the update information on the genetic diversity of the ten known BLV genotypes.

505 BLV isolates were generated during the analysis of restriction mappings of the env gene locus according to 5 restriction enzymes. The interpretation of their env-PCR-RFLP profiles actually reflects the strategy of PCR-RFLP genotyping of BLV in accordance with its phylogenetic classification. The data are represented in Table 6.

 

 

Table 5. Comparison of in silico data of PCR-RFLP (genotyping according to H. Fechner et al., 1997) and the phylogenetic analysis of a fragment of the BLV env gene

 

PCRRFLP genotyping

PCR product

(bp)

RFLP fragments (bp)

BLV genotypes

N

BamHI

BclI

BglI

HaeIII

PvuII

1

2

3

4

5

6

7

8

9

10

Variant group

A

444

444

225/219

328/116

198/94/87/32/27/6

280/164

142

142

198/121/87/32/6

444

198/119/94/27/6

285/94/32/27/6

B

444

316/128

219/121/104

328/116

285/94/32/27/6

444

8

6

1

15

444

C

444

316/128

225/219

328/116

198/94/87/32/27/6

444

56

4

28

70

19

177

285/94/32/21/6/6

285/94/32/27/6

444

198/87/49/45/32/27/6

198/119/94/27/6

D

444

444

225/219

328/116

198/94/87/32/27/6

444

42

1

2

45

E

444

316/128

225/219

328/116

225/94/8732/6

444

21

21

F

444

316/128

225/219

328/116

198/94/87/32/27/6

280/164

36

9

3

48

G

444

316/128

225/219

444

312/94/32/6

444

1

1

?

444

316/128

444

328/116

198/94/87/32/27/6

444

1

2

3

?

444

316/128

225/191/28

328/116

198/119/94/27/6

444

1

1

?

444

444

225/219

444

285/94/32/27/6

444

1

3

4

?

444

316/128

444

328/116

198/94/87/32/27/6

280/164

1

1

?

444

316/128

219/189/36

328/116

198/94/87/32/27/6

280/164

1

1

?

444

316/128

225/219

444

285/94/32/27/6

280/164

1

1

?

444

316/128

444

328/116

198/87/49/45/32/27/6

444

1

1

?

444

444

444

328/116

198/94/87/32/27/6

280/164

1

1

?

444

253/191

225/219

328/116

198/94/87/32/27/6

280/164

1

1

?

444

444

219/121/104

328/116

285/94/32/27/6

280/164

4

4

?

444

316/128

444

328/116

198/121/87/32/6

444

1

1

?

444

316/128

444

444

198/94/87/32/27/6

444

11

11

?

444

242/128/74

225/219

328/116

198/94/87/32/27/6

444

1

1

?

444

316/128

444

444

198/121/87/32/6

444

1

1

?

444

444

225/219

328/116

198/94/81/32/27/6/6

444

6

6

?

444

444

444

328/116

198/94/81/32/27/6/6

444

7

7

?

444

444

225/219

444

279/94/32/27/6/6

444

11

11

Legend. N is the number of analysed BLV isolates with an established PCR-RFLP profile; “?” – an unclassifiable BLV taxon.

 

 

Table 6. An improved strategy for PCR-RFLP-genotyping of BLV, consistent with the phylogenetic classification

 

G

BLV isolate

GenBank

A/N

PCR product

(bp)

RFLP fragments (bp)

C

N

PvuII

SspI

HphI

HaeIII

BstYI

1

AL–63

FJ808571

444

444

399/45

224/220

198/94/87/32/27/6

198/128/118

1

56

1

Cow 527

AF007764

444

444

399/45

224/220

285/94/32/27/6

198/128/118

2

8

1

23

U87873

444

444

399/45

224/220

312/94/32/6

198/128/118

3

1

1

AL–2106

FJ808578

444

444

399/45

224/220

198/94/87/32/27/6

246/198

4

42

1

UruC06II

FM955558

444

444

399/45

224/220

285/94/32/27/6

246/198

5

1

1

VdM

M35239

444

444

399/45

224/181/39

198/94/87/32/27/6

316/128

6

1

1

Kurdistan

EU266062

444

444

399/45

220/196/28

198/119/94/27/6

198/128/118

7

1

2

AL–164

FJ808574

444

280/164

399/45

224/220

198/94/87/32/27/6

198/128/118

8

34

2

PL–4960

FJ808590

444

280/164

399/45

224/220

198/87/49/45/32/27/6

198/128/118

9

1

2

ARGSF8

AF485773

444

280/164

399/45

444

198/94/87/32/27/6

198/128/118

10

1

2

AL–1453

FJ808577

444

280/164

444

224/220

198/94/87/32/27/6

198/128/118

11

1

3

USCA–1

EF065647

444

444

399/45

444

285/94/32/21/6/6

198/128/96/22

12

1

3

USCA–2

EF065648

444

444

399/45

444

285/94/32/27/6

198/128/96/22

13

2

3

JPFU

EF065650

444

444

399/45

444

285/94/32/27/6

198/128/118

14

1

4

BG

EF065638

444

280/164

399/45

224/220

198/94/87/32/27/6

444

15

115

4

3

U87872

444

444

399/45

224/220

198/94/87/32/27/6

444

16

1

4

1S–c16

JQ353652

444

280/164

399/45

444

198/94/87/32/27/6

444

17

16

4

N023

KC867149

444

280.164

399.45

224.220

198/94/87/32/27/6

253/191

18

1

4

1_BY

HQ902258

444

280/164

444

224/220

198/94/87/32/27/6

444

19

7

4

N034

KC886611