Introduction

Bovine leukemia virus (BLV) is a member of the genus Deltaretrovirus in the family Retroviridae [1], and isolates of this virus have been used as a model for research on the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1) [2]. BLV is considered the etiologic agent of enzootic bovine leucosis (EBL), and it infects cattle worldwide, making a severe economic impact on the beef and dairy cattle industries [3]. Most BLV-infected cattle are asymptomatic; however, approximately one-third of infected animals develop persistent lymphocytosis (PL) characterized by the polyclonal proliferation of CD5+ B lymphocytes [3]. Only 1-5% of BLV-infected cattle show malignant monoclonal B-cell lymphosarcoma (LS) [4,5,6]. The symptoms/signs of BLV-infected cattle also include reduced milk production, weakness, digestive disturbance, loss of appetite, weight loss or general debility, and various neurological manifestations [7]. BLV infections are often overlooked because they are asymptomatic, resulting in a reservoir of infection for both horizontal and vertical transmission [8, 9].

The BLV genome consists of 8714 nucleotides and includes the essential genes gag, pro, pol, and env, which encode structural proteins and enzymes, the regulatory genes tax and rex, and the accessory genes R3 and G4, and two identical long terminal repeats (LTRs) [10, 11]. Similar to other members of the family Retroviridae, the BLV env gene, encodes the envelope glycoprotein precursor pr72env, which is important for viral infectivity and syncytium formation [12,13,14]. The env gene also encodes a mature surface glycoprotein (gp51) and a transmembrane protein (gp30) [13]. The glycoprotein gp51 plays a crucial role in the viral life cycle [15, 16]. It influences the capacity of BLV to enter cells and has been identified as a target of specific neutralizing antibodies [16, 17]. Three conformational epitopes are present in the N-terminal half of BLV gp51, F, G, and H [18], which are involved in viral infectivity and syncytium formation, whereas the C-terminal half contains the linear epitopes A, B, D, and E [18]. Therefore, the gp51 region has been widely used for BLV genotyping and phylogenetic studies to identify viral strains isolated in recent years [7, 19,20,21]. To date, at least ten genotypes of BLV have been identified based on the genetic characteristics of the envelope glycoprotein [7].

BLV has been detected worldwide in cattle, with different levels of prevalence and genotype distribution in different countries [19, 21]. In the past five years, many countries have reported BLV infection in cattle. The range of BLV prevalence is 5.3% - 87.8% in Thailand [20], 4.8% - 9.7% in the Philippines [5], 9.1% in Myanmar [7], and 42.3% - 77.4% in South America [21], with genotypes 1-8 documented, in addition to the novel genotypes 9 and 10, which were also discovered in South America [21] and Myanmar [7]. Previous studies have shown that the distribution of BLV genotypes is not always consistent with the country of origin; however, there is a general correlation between genotype distribution and geographic region [7, 21, 22]. In northern and northeastern China, the seroprevalence of BLV has been reported to be 18.29% in dairy and beef cattle [23] and 21.09% in yaks (Bos grunniens) in Gansu Province [24]. However, more information is needed on BLV genotypes in China, particularly regarding yaks on the Qinghai-Tibet Plateau.

Yaks are a unique bovine species that live at high altitudes. They are a valuable, semi-wild animal species of which 95% are distributed in the territory of China. Special white yaks live in Tianzhu City, Gansu Province [24]. Since their domestication at least 5000 years ago by ancestors of the present-day Chinese Tibetan people, yaks have lived exclusively in the cold highlands surrounding the Qinghai-Tibet Plateau (altitudes < 3000 m, average annual temperature < 0 °C) [25], including in the provinces of Qinghai, Gansu and Sichuan in northwestern and southwestern China. Because they live in a relatively secluded and very cold geographic region, only a few pathogens have been discovered in yaks [26, 27]. We have been involved in the surveillance of yak infections in recent years [25, 26] in a study that contributes to the health of animals and the Chinese Tibetan people.

In this study, we investigated the distribution of BLV strains in domestic yaks on the Qinghai-Tibet Plateau of China based on the amplification of BLV LTRs using a combination of BLV-CoCoMoqPCR-2 and nested PCR. The partial and full-length env-gp51 sequences of 14 different Chinese BLV strains in yaks were used for phylogenetic analysis and compared with isolates from other geographical locations worldwide. The nearly full-length genome sequences of six Chinese BLV strains were obtained, and their genetic variability and genotypes were also analysed and compared with those of 16 whole BLV genome sequences from the NCBI database. This study is the first to identity the BLV genotypes in yaks of China.

Materials and methods

Experimental sample collection and extraction of genomic DNA

Blood samples were collected from 798 apparently healthy domestic yaks from 27 different farms in three provinces between March and December 2016 (Fig. 1 and Table 1). Farms located in the primary domestic-yak-raising areas were chosen for sampling in each province. The yaks were divided by age into ≤ 1 year and > 1 year and by species into white yak and black yak (Table 1). Genomic DNA was extracted from 50 μL of whole blood from each sample, using an E-Z 96® Blood DNA Kit (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. The extracted DNA was stored at – 20 °C until required for BLV detection.

Fig. 1
figure 1

Map of China showing the number and species of yak from provinces around the Qinghai-Tibet Plateau included in this study. The three provinces in which sampling was performed, yak species and occurrence of BLV genotypes are indicated in the figure. “n” indicates the total number of samples in each province or for each species, and BLV(+) % indicates the BLV prevalence rate

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Table 1 Results of BLV detection by nested PCR for three provinces on the Qinghai Tibetan Plateau, China
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Detection of BLV provirus by nested PCR targeting LTR regions

BLV provirus was detected by amplifying the BLV LTR regions in each DNA sample using nested PCR as described previously [5, 7, 28]. The first PCR amplification was performed with primers BLTR256F and BLTR453R (Fig. 2 and Table 2). As an internal control, the BoLA-DRA gene was amplified using primers BDRA488F and BDRA1145R, and in the nested PCR, using primers BLTR306F and BLTR408R. The same master mixes and thermal cycler conditions were used for both PCR and nested PCR. The reactions consisted of 5 picomoles of each primer, 10 μL of 2× PCR mix (Omega), and 6 μL of nuclease-free water, with 3 μL of DNA added to the PCR mixture and 3 μL of PCR product added to the nested PCR mixture. The final volume was 20 μL. The thermal cycle reactions were conducted in a thermocycler (Bio-Rad, USA) with initial denaturation at 94 °C for 5 min, followed by 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 90 s. The last cycle was run at 72 °C for 5 min. PCR products were confirmed by electrophoresis in a 1.5% agarose gel stained with ethidium bromide and were visualized under ultraviolet light. Sterilized water was used as the PCR negative control.

Fig. 2
figure 2

Strategies and primers used to detect yak BLV and amplify env genes and full-length BLV genomes

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Table 2 Primers used for detection and full-length genome amplification of BLV in yaks
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Amplification and sequencing of partial BLV env-gp51 and complete env genes

Samples that were BLV positive by nested PCR targeting LTR regions were used for amplification of partial BLV env-gp51 and complete env genes, which were also amplified by nested PCR using primers P3 and PBLV-gp51 (Fig. 2 and Table 2). The reaction mixture contained 7 μL (initial PCR) or 6 μL (second PCR) of distilled water, 10 μL of 2× PCR mix, 0.5 μL of each primer, and 2 μL of DNA (initial PCR) or initial PCR product (second PCR) in a final volume of 20 μL. Conditions for PCR amplification were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 35 s, annealing at 58 °C for 30 s, and extension at 72 °C for 90 s (initial PCR) or 30 s (second PCR). The last cycle was run at 72 °C for 5 min. PCR with the external primers resulted in amplification of a 1810-bp DNA fragment that contained the complete env gene, and the internal primers amplified a 497-bp fragment of the gp51 region of the env gene.

Positive PCR products from each of the two rounds were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany) and sequenced (TaKaRa, Dalian, China). The sequences included a 1810-bp portion of the env gene and a 497-bp portion of the env-gp51 gene, corresponding to nucleotide positions 4612-6421 and 5090-5589 of the proviral DNA sequence of bovine leukemia virus, strain par62 and L1, respectively (GenBank accession numbers LC080656 and LC154848) [7, 21]. Editing, alignment, and identification of nucleotide sequences were performed using MEGA 7.1 software [29].

Amplification and sequencing of the whole BLV provirus genome

Because the strains all showed high sequence similarity to one another based on analysis of the partial BLV env-gp51 gene and the complete env gene, three strains of each genotype were selected randomly for subsequent sequencing. Four overlapping genomic fragments covering the complete BLV genome sequence were obtained by PCR amplification from six Chinese yak strains using the primers listed in Table 2 and Fig. 2. The final reaction mixture (25 μL) contained 12.5 μL of 2× PCR mix, 1 μL of each primer (each at 10 pmol), 3 μL of DNA template, and 7.5 μL of distilled water. PCR amplification was performed as follows: 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s per kilobase. The last cycle was run at 72 °C for 10 min. The four different BLV provirus genome PCR amplicons from each individual were purified using a QIAquick Gel Extraction Kit (QIAGEN, Germany) and sequenced (TaKaRa, Dalian, China). The sequences were assembled and edited using MEGA 7.1 [29] and DNAMAN 9.0 software to produce the final sequences of the viral genomes. The genome sequences included the essential structural and enzymatic gag, pro, pol, and env genes, regulatory genes tax and rex, and the accessory genes R3 and G4. Only two nucleotides were lacking from each of the identical long terminal repeats (LTRs), which were located at the initiation and termination sites and did not influence the analysis of the structure and function of the BLV genome.

Phylogenetic analysis of the partial BLV env-gp51 gene, the complete env gene, and the whole BLV provirus genome

The 16 different partial BLV env-gp51 sequences and 16 complete env sequences from all positive samples from China were, aligned with 71 partial BLV env-gp51 sequences and 26 complete env sequences, respectively, from GenBank, which were representative of the ten known BLV genotypes, using MEGA 7.1 software [29, 30]. Phylogenetic trees were constructed using the maximum-likelihood (ML) algorithm with the K2+G model of nucleotide substitution in MEGA 7.1. The reliability of the phylogenetic relationships was evaluated by nonparametric bootstrap analysis with 1000 replicates. The six complete genome sequences (8710 bp) of Chinese BLV strains were also aligned with 16 previously determined complete BLV sequences. Phylogenetic analysis of a 497-bp portion of BLV env-gp51, the complete env gene, and the whole BLV provirus genome was conducted using MEGA 7.1 [29]. Prediction of the protein sequences of the partial BLV env-gp51 genes through translation of nucleotide sequences to amino acid sequences was also performed using MEGA.

Results

The prevalence of BLV infection in domestic yaks on the Qinghai-Tibet Plateau of China

Blood samples from 798 yaks were tested for BLV by nested PCR targeting the LTR regions. In Gansu Province, the blood samples of yaks were collected from two different species in two different cities, Tianzhu and Gannan. Sixteen of 87 white yaks ≤ 1 year old (18.39%) and 23 of 119 white yaks > 1 year old (19.32%) tested positive for BLV provirus (Fig. 1 and Table. 1). In Gannan, the positive rates of BLV in black yaks were 17.11% (13/76) for yaks ≤ 1 year old and 20.93% (18/86) for yaks > 1 year old. Samples from 14 of 111 black yaks ≤ 1 year old (12.61%) and 21 of 125 black yaks > 1 year old (16.80%) from Qinghai Province were BLV positive. Of 194 samples collected in Sichuan, samples from 12 yaks ≤ 1 year old (12/92; 13.04%) and 17 yaks > 1 year old (17/102; 16.67%) were positive for BLV provirus.

Phylogenetic analysis and genotyping

In recent phylogenetic studies of the env gene of BLV strains isolated from cattle, this virus was classified into ten genotypes [5, 7, 19,20,21, 24]. Therefore, to gain insight into the number and frequency of genotypes of BLV strains in China, 16 different partial BLV env-gp51 sequences and 16 complete env sequences from all positive samples in China were aligned with 71 partial BLV env-gp51 sequences and 26 complete env sequences, respectively, from GenBank. As shown in Figs. 3 and 4, BLVs in this study were classified into two closely related genotypes, genotype 10 and genotype 6. Both genotypes were detected in black and white yak samples from Gansu and Qinghai provinces; however, only genotype 10 was found from yaks in Sichuan Province. Notably, in Gansu and Qinghai provinces, genotype 10 was less prevalent than genotype 6 (Table 2).

Fig. 3
figure 3

Phylogenetic tree of partial BLV env sequences from different geographical locations worldwide. The phylogenetic tree was constructed from 16 distinct 497-bp BLV env sequences generated in this study and 71 sequences from known BLV strains, representing the ten different BLV genotypes. Bootstrap values expressed as percentages of 1000 replications are given at the branch points. The Chinese BLV strains identified in this study are indicated by GenBank accession numbers and breeds and strains. Other isolates are indicated by accession number and country of origin. The 16 newly identified yak BLV strains of genotype 10 and genotype 6 described in the present study (GenBank accession numbers MF574053-MF574068) are indicated by “●” and“▼”, respectively. Genotypes are indicated by numbers around the circumference of the figure. Genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 are shown in dark green, pink, mazarine, violet, green, pastel blue, orange, black, black-yellow and red, respectively. The scale bar indicates nucleotide substitutions per site

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Fig. 4
figure 4

Maximum-likelihood (ML) phylogenetic tree showing complete BLV env sequences from different geographical locations worldwide. The ML phylogenetic tree was constructed from complete (1448 bp) BLV env sequences from Chinese BLV strains (submitted to the GenBank nucleotide sequence database and assigned accession numbers MF574053-MF574068) and 26 sequences from known BLV strains (representing ten different BLV genotypes from different locations). These reference sequences were obtained from the GenBank nucleotide sequence database. The Chinese BLV strains identified in this study are indicated by GenBank accession numbers and breeds and strains. Other isolates are indicated by accession number and country of origin. The 16 newly identified yak BLV strains of genotype10 and genotype 6 described in the present study are indicated by “●” and“▼”, respectively. Genotypes are indicated by numbers to the right of the figure with different colours. Genotype 10 (G-10) is highlighted in grey. The bar at the bottom of the figure denotes evolutionary distance

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Sequence comparison of BLV strains from China

To compare the sequences and determine the genotypes of the BLV strains, 16 amplified fragments including full-length env genomes and the nearly full-length BLV genomes of six strains were sequenced and deposited in the GenBank database (accession numbers MF574053-MF574068 and MF580990-MF580995). These sequences were used to construct an ML phylogenetic tree (Figs. 4 and 5). The nucleotide sequence similarity ranged from 98.6% to 99.8% among the full-length env genomes of the eight strains and the nearly full-length BLV genome sequences of the three strains in genotype 6 that were discovered in this study, and the similarity with corresponding genotype strains whose sequences were obtained from GenBank was from 97.4% to 99.8%. The nucleotide sequence similarity of BLV strains of genotype 10 ranged from 97.5% to 99.8%, and the similarity with strains of the corresponding genotype obtained from GenBank ranged from 95.7% to 99.0%.

Fig. 5
figure 5

Maximum-likelihood (ML) phylogenetic tree constructed from complete BLV genomic sequences. The ML phylogenetic tree was constructed using the complete BLV genomic sequences from six Chinese BLV strains (submitted to the GenBank nucleotide sequence database and assigned accession numbers MF580990-MF580995) and 16 reference sequences obtained from the GenBank nucleotide sequence database. One thousand replications were performed to calculate bootstrap values (indicated on the tree). The strains identified in this study are indicated by GenBank accession numbers and breeds and strains. Reference sequences are indicated by accession number and country of origin. The 16 newly identified yak BLV strains of genotype 10 and genotype 6 described in the present study are indicated by “●” and“▼”, respectively. Genotypes are indicated by numbers to the right of the figure with different colours. The bar at the bottom of the figure denotes evolutionary distance

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Nucleotide and amino acid substitutions in BLV env-gp51 from strains isolated in yak of China

Six nucleotide sequences of each genotype were selected and aligned with that of a reference sequence of the same genotype obtained from GenBank (Figs. 6A and 7A). The Chinese strains shared 16 nucleotide substitutions (nucleotides 359, 442, 481, 484, 493, 517, 542, 545, 548, 558, 576, 577, 600, 618, 630 and 717) with the genotype-10 strains from Myanmar, although some contained 14 nucleotide mutations (Fig. 7A). In genotype-6, 13 nucleotide substitutions (nucleotides 306, 390, 420, 439, 457, 465, 507, 516, 570, 618, 615, 717 and 727) were shared between Chinese strains and those from Paraguay, although these nucleotide mutations only occurred in some of the strains (Fig. 6A).

Fig. 6
figure 6

Alignment of a partial nucleotide sequences and deduced amino acid sequences of the BLV env gene from strains of genotype 10 in China. Alignments were performed for eight typical nucleotide sequences (A) and eight unique deduced amino acid sequences (B) of the env gene from 134 Chinese BLV strains. Chinese BLV strains are identified by the sample ID at the left side of the figure. The numbers indicate the location of substitutions. The first, second and third neutralizing domains (ND) and other epitopes are shown at the top of the alignment in B. Numbers above the sequences are amino acid residue numbers that indicate the start and end of each domain. Genotypes are indicated by the black bars at the far left of the figure. The strain with accession number LC154849 from Myanmar was used as a reference

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Fig. 7
figure 7

Alignment of a partial nucleotide sequence and deduced amino acid sequence of the BLV env gene from strains of genotype 6 in China. Alignments were performed for eight typical nucleotide sequences (A) and eight unique deduced amino acid sequences (B) of the env gene from 134 Chinese BLV strains. Chinese BLV strains are identified by the sample ID at the left side of the figure. The numbers indicate the locations of substitutions. The first, second and third neutralizing domains (ND) and other epitopes are shown at the top of the alignment in B. Numbers above the sequences are amino acid residue numbers that indicate the start and end of each domain. Genotypes are indicated by the black bars at the far left of the figure. The strain with accession number LC080656 from Paraguay was used as a reference

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To identify the amino acid changes in the env proteins of the Chinese BLV strains, we aligned the deduced amino acid sequences of the eight partial env-gp51 sequences with those of representative genotype-10 sequences from Myanmar and genotype-6 sequences from Paraguay. As shown in Figs. 6B and 7B, amino acid substitutions occurred primarily in the middle region of gp51 of some of Chinese BLV strains. As expected, the Chinese strains had 10 amino acid substitutions that aligned with those of the Myanmar BLV strains, which were located in functional domains and included substitutions of histidine to arginine at residue 121 in the G-epitope region and histidine to lysine at residue 147 within the second neutralizing domain (2nd ND) region, eight substitutions (glutamic acid 160 glutamine, isoleucine 161 leucine, tyrosine 164 asparagine, arginine 182 glutamine, proline 183 histidine, aspartic acid 184 valine, glutamine 187 histidine, and proline 194 alanine) within the CD8+ T-cell epitope and E-epitope regions, and substitution of glutamic acid to aspartic acid at residue 224 in the third neutralizing domain (3rd ND) region (Fig. 7B). Furthermore, a substitution of arginine to glutamine at residue 182 occurred in all Chinese genotype-10 strains when compared with BLV strains from Myanmar. By contrast, alignment with sequences from Paraguay revealed only two amino acid substitutions in the genotype 6 strains: histidine to tyrosine at residue 152 and glutamic to aspartic acid within the 3rd ND region (Fig. 6B).

Discussion

Four primary conclusions were reached based on the results of this study. First, the comprehensive investigation demonstrated widespread distribution of BLV in domestic yaks in provinces surrounding the Qinghai-Tibet Plateau of China. As determined by nested PCR targeting the LTR regions, a total of 798 samples collected from different provinces and cities located on the Qinghai-Tibet Plateau of China showed a relatively high rate of BLV infection compared with the infection rates in other countries [5, 7]. Second and most importantly, the genotypes of BLV in yaks were identified for first the time in China, and a phylogenetic analysis based on partial and complete env-gp51 sequences revealed that Chinese BLV strains clustered with genotype 10 and genotype 6 of the ten distinct BLV genotypes found worldwide [7, 20,21,22, 31, 32]. Third, an ML phylogenetic tree based on 497 bp of the BLV env sequence clearly showed that Chinese BLV strains in yaks belonged to other genotypes, including a new genotype found in Southeast Asia (Myanmar and Thailand) (Fig. 3), which is very close to China [7, 20]. Finally, several nucleotide and amino acid substitutions were discovered in the full BLV genome sequences and in the gp51 gene sequences of the Chinese strains, particularly for genotype 10. Additionally, most of the substitutions were observed in structural genes, such as the CD8+ T-cell epitope, the second ND and third ND, and the G and E epitopes, and the substitutions varied according to genotype, which was similar to the genetic characteristics of BLV in the Philippines [5].

The BLV infection rate was 16.79% (134/798) in the yaks surrounding the Qinghai-Tibet Plateau of China as determined by PCR. In neighbouring countries, the percentage of BLV infection is 5.3% to 87.8% in Thailand [20], 9.1% in Myanmar[7], and 4.8% to 9.7% in Philippines [5]. In the present study, the percentage of BLV infection in Chinese yaks was lower than that in cattle of other countries such as Korea (35%) [33] and Tanzania (36%) [34] but higher in Chinese yaks than the 9.1% in Myanmar and the 4.8% to 9.7% in the Philippines. However, the prevalence of BLV in yaks was significantly lower than that in cattle in South America (42.3% to 77.4%) [21]. In China, the seroprevalence of BLV is 18.29% in dairy and beef cattle [23], whereas in Gansu Province, the prevalence in yaks was 21.09%. Furthermore, 24.26% of black yaks and 19.10% white yaks tested positive in an earlier study [24], which were higher values than those we reported in this study. The percentage of BLV infection in black and white yaks was 18.93% and 19.14%, respectively, and the difference was not significant, which is consistent with the results of serological detection [24]. Based on the methods of nested PCR in this study and serological detection in a previous study [24], the differences of BLV prevalence between yaks > 1 year old and yaks ≤ 1 year old were also not significant. Another notable result was that Gansu showed the highest prevalence of BLV infection, including 18.93% in white yaks from Tianzhu City and 19.14% in black yaks from Gannan City. The prevalence of BLV in black yaks from Qinghai and Sichuan was 14.83% and 14.94%, respectively. These results indicate that age and breed are not risk factors for the presence of BLV in yaks; however, differences in the percentage of BLV infection are likely to occur between provinces and locations within the same country.

The most interesting data in this study were from the molecular characterization of BLV in yaks confirmed by phylogenetic analysis using six new and 16 previously reported BLV whole genome sequences (Fig. 5), which were consistent with those of phylogenetic analysis of 16 partial env-gp51 sequences and full-length env sequences obtained by the ML method (Figs. 3 and 4). Two genotypes (genotype 10 and genotype 6) were identified in BLV strains from yaks in China. The presence of more than one genotype in the same herd has been observed previously in open herds [35]. The genotype 10 BLV strains in China and the strains from Myanmar, with the Chinese BLV strains identified in samples located close to one another, were detected in all three provinces in this study. By contrast, genotype 6 BLV strains were detected in Gansu and Qinghai provinces but were not found in Sichuan Province, and these strains were also located close to the strains from Thailand. Genotype 10 is a new genotype of BLV that was discovered in recent years [7, 20], and it was detected in all provinces surrounding the Qinghai-Tibet Plateau of China. Collectively, our study is the first to show that BLV strains isolated from yaks in China were most similar to BLV strains from Myanmar and Thailand and that the infection of yaks with BLV might have occurred in recent years.

Our analysis of partial of BLV env-gp51 sequences identified 16 nucleotide substitutions in genotype 10; six of which were silent substitutions and 11 of which resulted in amino acid substitutions. Thirteen nucleotide substitutions were identified in genotype 6 among the Chinese strains, two of which resulted in amino acid substitutions. More substitutions were found in genotype 10 than in genotype 6, and these substitutions also occurred in the same genotype found in this study. This result indicated that the sequences of the genotype-6 BLV strains in yaks were more conserved than those of the genotype-10 BLV strains. Furthermore, almost all substitutions were located in functional epitopes or NDs, which is consistent with a previous finding that most substitutions in env-gp51 are found within epitopes rather than at random locations [17, 22, 33, 36, 37]. Amino acid substitutions in the second ND could affect the interaction between gp51 and a receptor expressed on host-cell membranes, suggesting that these substitutions could affect viral fusion and infectivity in vivo [38]. The CD4+ epitope, CD8+ epitope, E epitope and G epitope are conformational epitopes and are targets for monoclonal antibodies that induce neutralization and inhibit syncytium formation [38, 39]. Therefore, the biological effects of these substitutions should be investigated.

The present study reveals the wide prevalence of BLV infection in yaks surrounding the Qinghai-Tibet Plateau of China, and the molecular characteristics of BLV in yaks were determined for the first time. BLV genotypes 10 and 6 were prevalent in domestic yaks in China. These results will be very important for clarifying the source, circulation pattern, zoonotic potential, and public health risk of this virus. The mechanisms of the pathogenicity, transmission, evolution, and persistence of this virus also require urgent clarification.